EP1485128A2 - Nanostructures contenant des unites d'assemblage d'anticorps - Google Patents

Nanostructures contenant des unites d'assemblage d'anticorps

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Publication number
EP1485128A2
EP1485128A2 EP03713600A EP03713600A EP1485128A2 EP 1485128 A2 EP1485128 A2 EP 1485128A2 EP 03713600 A EP03713600 A EP 03713600A EP 03713600 A EP03713600 A EP 03713600A EP 1485128 A2 EP1485128 A2 EP 1485128A2
Authority
EP
European Patent Office
Prior art keywords
antibody
assembly
nanostructure
joining
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03713600A
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German (de)
English (en)
Other versions
EP1485128A4 (fr
Inventor
Lee Makowski
Mark K. Williams
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanoframes Inc
Original Assignee
Nanoframes Inc
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Filing date
Publication date
Application filed by Nanoframes Inc filed Critical Nanoframes Inc
Publication of EP1485128A2 publication Critical patent/EP1485128A2/fr
Publication of EP1485128A4 publication Critical patent/EP1485128A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • B82B3/0009—Forming specific nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to methods for the assembly of nanostructures containing antibody or antibody fragment assembly units for use in the construction of such nanostructures, and to nanostructures containing antibody or antibody assembly units.
  • Nanostructures are structures with individual components having one or more characteristic dimensions in the nanometer range (from about 1-100 nm).
  • the advantages of assembling structures in which components have physical dimensions in the nanometer range have been discussed and speculated upon by scientists for over forty years. The advantages of these structures were first pointed out by Feynman (1959, There's Plenty of Room at the Bottom, An invitation to Enter a New Field of Physics (lecture), December 29, 1959, American Physical Society, California Institute of Technology, reprinted in Engineering and Science, February 1960, California Institute of Technology, Pasadena, CA) and greatly expanded on by Drexler (1986, Engines of Creation, Garden City, N.Y.: Anchor Press/Doubleday). These scientists envisioned enormous utility in the creation of architectures with very small characteristic dimensions.
  • the physical and chemical attributes of a nanostructure depend on the building blocks from which it is made. For example, the size of these building blocks, and the angles at which they join plays an important role in determining the properties of the nanostructure, and the positions in which functional elements can be placed.
  • the art provides numerous examples of different types of materials which can be used in nanostructures, including DNA (US Patents Nos. 5,468,851, 5,948,897 and 6,072,044; WO 01/00876), bacteriophage T even tail fibers (US Patents Nos.
  • the present invention provides nanostructures formed from a plurality of species of assembly units. With some exceptions, such as capping units, these assembly units comprise a plurality of different joining elements.
  • the nanostructure includes at least one species of assembly unit in which at least one joining, structural or functional element comprises an antibody or antibody fragment.
  • the antibody assembly units may have one or more antibody or antibody fragment elements, and in addition the antibody assembly units may contain other, non-antibody, structural, functional and joining elements.
  • the nanostructure of the invention is suitably prepared using a staged assembly method, i this method, a nanostructure intermediate comprising at least one unbound joining element is contacted with an assembly unit comprising a plurality of different joining elements, wherein:
  • none of the j oining elements of said plurality of different j oining elements can interact with itself or with another joining element of said plurality, and
  • the assembly unit in at least one cycle comprises an antibody assembly unit.
  • FIG. 1 Diagram of an idiotope/anti-idiotope Fab-Fab interaction.
  • the diagram shows the ⁇ -carbon trace of two Fab fragments interacting through idiotopic/anti-idiotopic interactions (pdb entry 1CIC).
  • the heavy lines represent the heavy chains and the light lines represent the light chains of the Fab fragments.
  • Most of the idiotopic/anti-idiotopic protein binding interactions occur between the loops of the heavy chains contained in the complementarity determining region (CDR). In this case, the association between Fabs results in a nearly linear association.
  • CDR complementarity determining region
  • FIG. 2 Line drawing representing the three-dimensional structures of the ⁇ -carbon trace of a diabody (pdb entry 1LMK) (top) and a single chain Fv (scFv) antibody (pdb entry 2APA) (bottom).
  • scFv single chain Fv
  • pdb entry 2APA single chain Fv
  • heavy lines represent the heavy chain and the light lines represent the light chain.
  • the heavy lines represent both the heavy chain and light chain of one scFv, while the light lines represent both the heavy and light chain of the other scFv.
  • scFv constructs that have the heavy-light variable domains linked together by a longer peptide linkers form stable monomers.
  • scFv and diabodies, or binding derivatives or binding fragments thereof, can be used as the basic elements for the design of assembly units.
  • FIG. 3 Diagram of single-chain Fv fragments (scFv). The top half of the diagram shows monomeric, dimeric (diabody), trimeric (triabody) and tetrameric (tetrabody) associations among V H - linker -V L scFv fragments. The bottom half of the diagram shows such associations among V L - linker -V H scFv fragments. These associations between scFv domains are dependent upon the length of the peptide linker joining the V H and V L units. Longer peptide linkers (12-15 residues) favor monomeric formation, whereas shorter linkers (0-5 residues), favor multimeric structures.
  • V H and V L genes also affects multimer formation, activity and stability of the resultant scFv proteins.
  • This type of recombinant antibody represents one of the smallest functional antigen binding entities derived from an IgG and can be utilized as the structural and joining elements in assembly unit fabrication.
  • Unit 1 is an A x B diabody in which the V H and V L domains of A define a lysozyme isotopic antibody (D1.3) and in which the V H and V L domains of B define a virus neutralizing idiotopic antibody (730J .4).
  • the gene encoding V H A and V L B includes a hexahistidine tag, whereas the gene encoding V H B and V L A does not.
  • Unit 2 is B' x A' diabody in which the V H and V L domains of B' define a virus neutralizing idiotopic antibody (409.5.3) and in which the V H and V L domains of A' define a lysozyme isotopic antibody (E5.2).
  • the gene encoding V H B' and N L A' includes a hexahistidine tag, whereas the gene encoding V H A' and V L B' does not.
  • FIG. 5 Line drawing representing the ⁇ -carbon trace of an intact IgGl (Protein Data Bank (pdb) entry 1IGY) (Harris et al, 1998, Crystallographic structure of an intact IgGl monoclonal antibody, j. Mol. Biol. 275(5): 861-72). (For a description of the Protein Data Bank (pdb), see Berman et al, 2000, The Protein Data Bank, ⁇ ucl. Acids Res. 235-42; Saqi et al, 1994, PdbMotif ⁇ a tool for the automatic identification and display of motifs in protein structures, Comput. Appl. Biosci. 10(5): 545-46.) Thick lines represent the heavy chains and thin lines represent the light chains.
  • the Fv and C H 1 domains of the Fab fragment and the C H 2 and C H 3 domains of the Fc fragment are labeled.
  • Ball-and-stick modeling indicated by gray arrowheads, represent disulfide cysteine bonds. Clusters of disulfide bridging interactions occur in the flexible hinge region located between the Fab and Fc fragments. These interactions may aid in dimerization and provide structural integrity of this otherwise highly flexible region in the immunoglobulin. Drawing was created with the program SETOR (Evans, 1993, SETOR: Hardware lighted three- dimensional solid model representations of macromolecules, j. Mol. Graphics, 11: 134- 38).
  • FIG. 6 Line drawing representing the ⁇ -carbon trace of a Fab fragment that can be used as the structural element for design of an assembly unit (pdb entry ICIC ).
  • the heavy lines represent the heavy chain and the light lines represent the light chain.
  • the domains of the heavy chain (V H and C H 1) and the light chain (V L and C-J are labeled.
  • the flexible Fab "elbow” or bend region connecting the variable domains and constant domains. The Fab angle of the bend varies considerably (127-176°) even among members of the same antibody class.
  • FIG. 7 Schematic representation of various IgGs including monovalent, bivalent, monospecific and bispecific antibodies.
  • IgGs that are derived from a single cell line are homozygous for IgG.
  • the resulting IgGs are therefore bivalent-monospecific antibodies.
  • a hybrid hybridoma e.g., a quadroma, arises from a fusion cell line.
  • IgGs that are produced by hybrid hybridomas may be mixtures of heterologous bivalent-bispecific (e.g., heterologous-F(ab') 2 ) and homozygous bivalent-monospecific (e.g., F(ab') 2 ) IgG.
  • Hybrid hybridoma heterodimers therefore represent a source of bivalent-bispecific F(ab') 2 .
  • the intact IgG molecules or binding derivative or binding fragment thereof can be used as the basic elements for the design of assembly units.
  • FIG. 8 Schematic representation of an IgG molecule cleaved into its component fragments, F(ab') 2 and Fc, upon limited exposure to protease.
  • the hinge region containing several disulfide-bond interactions, helps maintain dimerization of the Fab fragments. Subsequent exposure of the F(ab') 2 to reducing conditions disrupts the hinge disulfide bridging interactions between the fragments to yield monomeric Fab.
  • Separate functional fragments of the IgG can be isolated (i.e., Fab fragments) for specific uses in the design of assembly units such as creating bivalent-bispecific heterologous F(ab') 2 by chemical cross-linking.
  • Leucine zipper motifs (depicted as elongated ovals) such as Jun-Fos or GCN4 (Kostelny et al, 1992, Formation of a bispecific antibody by the use of leucine zippers, J. Immunol. 148(5): 1547-53; de Kruif et al, 1996, Leucine zipper, dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library, J. Biol. Chem.
  • FIG. 10 Diagram of ROP protein, a four-helix bundle.
  • FIG. 11 Staged assembly of assembly units.
  • each step in the staged assembly will be carried out in a massively parallel fashion.
  • an initiator unit is immobilized on a solid substrate.
  • the initiator unit has a single joining element, h step 2, a second assembly unit is added.
  • the second unit has two non-complementary joining elements, so that the units will not self-associate in solution.
  • One of the joining elements on the second assembly unit is complementary to the joining element on the initiator unit. Unbound assembly units are washed away between each step (not shown).
  • the second assembly unit binds to the initiator unit, resulting in the formation of a nanostructure intermediate made up of two assembly units, h step 3, a third assembly unit is added.
  • This unit has two non-complementary joining elements, one of which is complementary to the only unpaired joining element on the nanostructure intermediate.
  • This unit also has a functional unit ("F3").
  • a fourth assembly unit with functional element "F4" and a fifth assembly unit with functional element "F5" are added in steps 4 and 5, respectively, in a manner exactly analogous to steps 2 and 3. hi each case, the choice of joining elements prevents more than one unit from being added at a time, and leads to a tightly controlled assembly of functional units in pre-designated positions.
  • FIG. 12 Generation of a nanostructure from subassemblies.
  • a nanostructure can be generated through the sequential addition of subassemblies, using steps analogous to those used for the addition of individual assembly units as illustrated above in FIG. 2.
  • the arrow indicates the addition of a subassembly to a growing nanostructure.
  • FIG. 13 A diagram illustrating the addition of protein units and inorganic elements to a nanostructure according to the staged assembly methods of the invention.
  • an initiator unit is bound to a solid substrate.
  • an assembly unit is bound specifically to the initiator unit.
  • an additional assembly unit is bound to the nanostructure undergoing assembly.
  • This assembly unit comprises an engineered binding site specific for a particular inorganic element.
  • the inorganic element (depicted as a cross- hatched oval) is added to the structure and bound by the engineered binding site.
  • Step 5 adds another assembly unit with a binding site engineered for specificity to a second type of inorganic element, and that second inorganic element (depicted as a hatched diamond) is added in step 6.
  • FIG. 14 Diagram of eleven steps of a staged assembly that utilizes four bispecific assembly units and one tetraspecific assembly unit to make a two-dimensional nanostructure.
  • FIGS. 15(A-B). Diagram of a staged assembly that utilizes nanostructure intermediates as subassemblies.
  • Steps 1-3 a nanostructure intermediate is constructed, two joining elements are capped and the nanostructure intermediate is released from the solid substrate, hi Step 5, the nanostructure intermediate from Step 3 is added to an assembly intermediate (shown in Step 4 attached to the solid substrate) as an intact subassembly.
  • FIGS. 16(AA-BF). Diagram of the sequence of the 32 steps used in the staged assembly of an exemplary cubic nanostructure.
  • the cubic nanostructure is assembled from assembly units comprising structural elements from engineered diabody and triabody fragments.
  • the joining elements of the assembly units are the multispecific binding domains from diabodies or triabodies. Seven complementary joining pairs are used: A and A', B and B', C and C, D and D', E and E', F and F, and G and G'.
  • the numbering (1-32) indicates the assembly unit added during each step.
  • Antibody Assembly Unit An assembly unit in which at least one joining element, structural element or functional element is an antibody or antibody fragment, or a binding derivative thereof.
  • the antibodies, binding derivatives or binding fragments may be of any class of immunoglobulin molecules, including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • Antibody Fragment A portion of an antibody with specific binding affinity for an epitope.
  • antibody fragments include, without limitation, Fab or F(ab') 2 antibody fragments, single-chain antibody fragments (scFvs), bispecific IgG, chimeric IgG or bispecific heterodimeric F(ab') 2 antibodies, diabodies or multimeric scFv fragments.
  • An assembly unit is an assemblage of atoms and/or molecules comprising structural elements, joining elements and/or functional elements. Preferably, an assembly unit is added to a nanostructure as a single unit through the formation of specific, non-covalent interactions.
  • An assembly unit may comprise two or more sub- assembly units.
  • An assembly unit may comprise one or more structural elements, and may further comprise one or more functional elements and/or one or more joining elements. If an assembly unit comprises a functional element, that functional element may be attached to or incorporated within a joining element or, in certain embodiments, a structural element.
  • Such an assembly unit which may comprise a structural element and one or a plurality of non-interacting joining elements, maybe, in certain embodiments, structurally rigid and have well-defined recognition and binding properties.
  • An initiator assembly unit is the first assembly unit incorporated into a nanostructure that is formed by the staged assembly method of the invention. It may be attached, by covalent or non-covalent interactions, to a solid substrate or other matrix as the first step in a staged assembly process.
  • An initiator assembly unit is also known as an "initiator unit.”
  • binding derivative of an antibody or antibody fragment is a derivative that exhibits the binding specificity of the antibody, antibody fragment, single-chain antibody fragment (scFv), etc., from which the binding derivative is derived.
  • a binding fragment of an antibody or antibody fragment is a fragment that exhibits the binding specificity of the antibody, antibody fragment, single-chain antibody fragment (scFv), etc., from which the binding fragment is derived.
  • Bottom-up assembly of a structure is formation of the structure through the joining together of substructures using, for example, self-assembly or staged assembly.
  • a capping unit is an assembly unit that comprises at most one joining element. Additional assembly units cannot be incorporated into the nanostructure through interactions with the capping unit once the capping unit has been incorporated into the nanostructure.
  • Derivatives of a protein of interest used in the methods of the mvention can be made by altering sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as the gene encoding the protein of interest may be used in the practice of the present invention. These include, but are not limited to, nucleotide sequences comprising all or portions of a gene, which is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • derivatives of a protein of interest include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a protein of interest including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • derivatives or analogs of antibodies include but are not limited to those molecules comprising regions that are substantially homologous to the antibody of interest or a bindmg fragment thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a sequence encoding the protein of interest, under highly stringent or moderately stringent conditions. Such highly or moderately stringent conditions are commonly known in the art.
  • First Assembly Unit, First Element For clarity, assembly units or elements are sometimes referred to using labels such as "first" or "second". This is purely a labeling convention and in no way indicates the position of the referenced assembly unit or element within the nanostructure.
  • a functional element is a moiety exhibiting any desirable physical, chemical or biological property that may be built into, bound or placed by specific covalent or non-covalent interactions, at well-defined sites in a nanostructure.
  • a functional element can be used to provide an attachment site for a moiety with a desirable physical, chemical, or biological property.
  • Examples of functional elements include, without limitation, a peptide, protein (e.g., enzyme), protein domain, small molecule, inorganic nanoparticle, atom, cluster of atoms, magnetic, photonic or electronic nanoparticles, or a marker such as a radioactive molecule, chromophore, fluorophore, chemiluminescent molecule, or enzymatic marker.
  • Such functional elements can also be used for cross-linking linear, one-dimensional nanostructures to form two-dimensional and three-dimensional nanostructures.
  • a joining element is a portion of an assembly unit that confers binding properties on the unit, including, but not limited to: binding domain, hapten, antigen, peptide, PNA, DNA, RNA, aptamer, polymer or other moiety, or combination thereof, that can interact through specific, non-covalent interactions, with another joining element.
  • Complementary joining elements are two joining elements that interact with one another through specific, non-covalent interactions.
  • Non-complementary joining elements are two joining elements that do not specifically interact with one another, nor demonstrate any tendency to specifically interact with one another.
  • a joining pair is two complementary joining elements.
  • Nanomaterial is a material made up of a crystalline, partially crystalline or non-crystalline assemblage of nanoparticles.
  • Nanoparticle A nanoparticle is an assemblage of atoms or molecules, bound together to form a structure with dimensions in the nanometer range (1-1000 nm). The particle may be homogeneous or heterogeneous. Nanoparticles that contain a single crystal domain are also called nanocrystals.
  • Nanostructure or Nanodevice A nanostructure or nanodevice is an assemblage of atoms and/or molecules comprising assembly units, i.e., structural, functional and/or joining elements, the elements having at least one characteristic length (dimension) in the nanometer range, in which the positions of the assembly units relative to each other are established in a defined geometry. The nanostructure or nanodevice may also have functional substituents attached to it to provide specific functionality.
  • Nanostructure intermediate is an intermediate substructure created during the assembly of a nanostructure to which additional assembly units can be added. In the final step, the intermediate and the nanostructure are the same.
  • Non-covalent Interaction Specific: A specific non-covalent interaction is, for example, an interaction that occurs between an assembly unit and a nanostructure intermediate.
  • Protein h this application, the term “protein” is used generically to referred to peptides, polypeptides and proteins comprising a plurality of amino acids, and is not intended to imply any minimum number of amino acids.
  • Removing of unbound assembly is accomplished when they are rendered unable to participate in further reactions with the growing nanostructure, whether or not they are physically removed.
  • Self-assembly is spontaneous organization of components into an ordered structure. Also known as auto-assembly.
  • Staged Assembly of a Nanostructure is a process for the assembly of a nanostructure wherein a series of assembly units are added in a pre-designated order, starting with an initiator unit that is typically immobilized on a solid matrix or substrate. Each step results in the creation of an intermediate substructure, referred to as the nanostructure intermediate, to which additional assembly units can then be added.
  • An assembly step comprises (i) a linking step, wherein an assembly unit is linked to an initiator unit or nanostructure intermediate through the incubation of the matrix or substrate with attached initiator unit or nanostructure intermediate in a solution comprising the next assembly units to be added; and (ii) a removal step, e.g., a washing step, in which excess assembly units are removed from the proximity of the intermediate structure or completed nanostructure. Staged assembly continues by repeating steps (i) and (ii) until all of the assembly units are incorporated into the nanostructure according to the desired design of the nanostructure. Assembly units bind to the initiator unit or nanostructure intermediate through the formation of specific, non-covalent bonds. The joining elements of the assembly units are chosen so that they attach only at pre-designated sites on the nanostructure intermediate.
  • the geometry of the assembly units, the structural elements, and the relative placement of joining elements and functional elements, and the sequence by which assembly units are added to the nanostructure are all designed so that functional units are placed at pre-designated positions relative to one another in the structure, thereby conferring a desired function on the completely assembled nanostructure.
  • Structural Element A structural element is a portion of an assembly unit that provides a structural or geometric linkage between joining elements, thereby providing a geometric linkage between adjoining assembly units. Structural elements provide the structural framework for the nanostructure of which they are a part.
  • a subassembly is an assemblage of atoms or molecules consisting of multiple assembly units bound together and capable of being added as a whole to an assembly intermediate (e.g., a nanostructure intermediate), hi many embodiments of the invention, structural elements also support the functional elements in the assembly unit.
  • assembly intermediate e.g., a nanostructure intermediate
  • Top-down assembly of a structure is formation of a structure through the processing of a larger initial structure using, for example, lithographic techniques.
  • the present invention provides a new class of assembly units that can be used in production of nanostructures.
  • These "antibody assembly units” contain at least one joining, structural or functional element that is an antibody or antibody fragment, hi addition, the assembly unit may contain structural elements and/or other joining and functional elements.
  • the present invention provides for the staged assembly of nanostructures that utilizes assembly units comprising chimeric antibodies or antibody fragments.
  • fusion or chimeric protein products comprising a desired protein (e.g., an IgG), fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)).
  • a desired protein e.g., an IgG
  • fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)
  • heterologous protein sequence of a different protein
  • Such chimeric protein products can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper reading frame, and expressing the chimeric product by methods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • Each IgG-derived antibody fragment preferably contains at least one monovalent and monospecific complementarity determining region (CDR) or joining element.
  • the CDR is preferably the site contained in each structure at which the highly specific intermolecular interaction can occur between the protein components.
  • Recombinantly engineered antibodies meet many of the basic criteria for use in the construction of assembly units for staged-assembly of nanostructures and are preferred sources of joining elements used for fabricating such nanostructures. Not only are such recombinant antibody binding domains structurally well characterized, they also have inherent binding specificities (joining elements) necessary for assembly unit addition.
  • the known three-dimensional structure of many recombinant engineered components can serve as a guide for design of structural modifications to the antibody fragment that will enable the insertion of peptides (for example, at the site of a surface loop) that will confer novel binding, structural or functional properties to the antibody fragment.
  • peptides for example, at the site of a surface loop
  • Methods 248(1-2): 183-94 that may be used, according to the methods of the invention, as sources of structural elements and joining elements.
  • Such multivalent, multispecific and multifunctional antibodies can be modified by the addition of functional groups for the construction of assembly units used for the fabrication of nanostructures as described herein.
  • joining elements are derived from antibodies, or binding derivatives or binding fragments thereof, and exhibit antigen- antibody interactions which are used in the formation of a joining pair.
  • Structural information is readily available for a variety of antibody-antigen complexes. Such structural information may be used to design joining elements for the fabrication of nanostructures according to the methods of the invention.
  • the variable domains of antibodies are designed to interact with specificity to an antigenic target. Their structure and stability are well-characterized in the art, and antibodies and antibody binding fragments may be engineered using methods well known in the art. Consequently, the variable domains of antibodies represent a class of molecules with great potential as joining elements for use as nanostructure assembly units. Such elements provide the basis for specific binding interactions between assembly units and initiators or nanostructure intermediates and are described herein.
  • This high specificity has been shown to correlate with the high complementarity between the antibody combining site and the antigenic determinant, i.e., the epitope or hapten.
  • This complementarity is defined by the antibody determinant face, defined as the complementarity determining region (CDR) and the antigenic determinant surface, which are in contact, so that the depressions in one are filled by the protrusions from the other.
  • CDR complementarity determining region
  • Complementarity also exists by physical and chemical properties such as opposed, oppositely charged side-chain interactions that form ionic bonds.
  • the specificity occurring between the CDR and the antigenic determinant surface can define one type or pair of non- complementary joining element interactions.
  • Antibodies or portions thereof used in the methods of the invention can be multispecific (i.e., demonstrate binding affinity towards more than one ligand) or monospecific (i.e., demonstrate binding affinity towards only one ligand). hi general, antibodies demonstrate bmding affinity in the 10 "1 to 10 "4 nM range or better (Padlan, 1994, Anatomy of the antibody molecule, Mol. Immunol. 31(3): 169-217).
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, the CDRs.
  • the Fv fragment contains six variable loop regions, three from the V L chain and three from the V H chain.
  • Each of the variable polypeptide loop regions contained in the variable chains display variability in residue sequence and length. Residues within this region are assigned either to hypervariable, complementarity-determining-regions (CDRs) or to non-hypervariable or framework regions (Wu et al, 1970, An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity, J. Exp.
  • variable loop regions define, almost entirely, the antigen- recognition site of the antibody.
  • CDR3s CDR3-L and CDR3-H
  • CDR3-L and CDR3-H are the most prominent in antibody-antigen recognition interactions and are the most variable in sequence and conformation.
  • the contributions from the CDR loops from both the V L and the V H chains on binding to antigen are relatively consistent.
  • Structural analyses of antibodies complexed with antigen have determined that approximately 41-44% of the interacting surface area is contributed by the light chain with the heavy chain contributing 56-59% (Davies et al, 1990, Antibody-antigen complexes, Annu. Rev. Biochem. 59: 439-73).
  • the overall number of residues that interact with the antigen is rather small.
  • a joining element comprises a recombinantly engineered antibody or binding derivative or binding fragment thereof.
  • recombinantly engineered antibodies known in the art that are multivalent, multispecific and/or multifunctional, and that are suitable as joining elements for use in the design of assembly units for staged assembly of nanostructures.
  • Such assembly units may either be unmodified or be modified as described herein, for use in the methods of the invention for fabrication of a desired nanostructure.
  • recombinantly engineered antibodies, or binding derivatives or binding fragments thereof, for use as joining elements include, but are not limited to:
  • bivalent, trivalent, mono-, bi-, or tri-specific antibodies with or without added functionalities such as IgGs derived from hybrid hybridomas, F(ab') 2 , diabodies, triabodies, tetrabodies, heterologous-F(ab') 2 , Fab-scFv fusions or F(ab') 2 -scFv fusions (Milstein and Cuello, 1983, Hybrid hybridomas and their use in immunohistochemistry, Nature 305(5934): 537-40; Neuberger et al, 1984, Recombinant antibodies possessing novel effector functions, Nature 312(5995): 604-08; Weiner, 1992, Bispecific IgG and IL-2 therapy of a syngeneic B-cell lymphoma in immunocompetent mice, Int.
  • functionalities such as IgGs derived from hybrid hybridomas, F(ab') 2 , diabodies, triabodies, tetrabodies, heterolog
  • tetravalent antibodies that are either, mono-, bi-, tri- or tetraspecific antibodies, with or without added functionalities, such as tetrabodies, Ig-G binding derivative-scFv fusions or IgG-scFv fusions (Pack et al, 1995, Tetravalent miniantibodies with high avidity assembling in Escherichia coli, J. Mol. Biol. 246(1): 28-34, Coloma and Morrison, 1997, Design and production of novel tetravalent bispecific antibodies, Nat. Biotechnol.
  • cytokine e.g., a BCDF (B-cell differentiation factor), a BCGF (B-cell growth factor), a motogenic cytokine, a chemotactic cytokine or chemokine, a CSF (colony stimulating factor), an angiogenesis factor, a TRF (T-cell replacing factor), an ADF (adult T-cell leukemia-derived factor), a PD-ECGF (platelet-derived endothelial cell growth factor), a neuroleukin, an interleukin, a lymphokine, a monokine, an interferon, etc.)(--?ee, e.g., Penichet and Morrison, 2001, Antibody-cytokine fusion proteins for the therapy of cancer, J.
  • a BCDF B-cell differentiation factor
  • BCGF B-cell growth factor
  • motogenic cytokine e.g., a chemotactic cytokine or chemokine
  • CSF colony stimulating factor
  • idiotope/anti-idiotope interactions are used to design joining elements for the construction of nanostructures according to the methods of the invention. Since antibodies can recognize virtually any antigen, they have the ability to recognize other antigenic determinants contained on other antibodies. The immune responses that arise from the potential antigenic determinants on antibodies are called "idiotopic" (Jerne, 1974, Towards a network theory of the immune system, Ann. Immunol. (Paris) 125C(l-2): 373-89; Davie et al, 1986, Structural correlates of idiotopes, Annu. Rev. Immunol. 4: 147-65). Idiotopes are the antigenic determinants unique to a particular antibody or group of antibodies.
  • Antibodies bearing idiotopes can react with antibodies that recognize the idiotope as antigen and are therefore termed "anti-idiotopic" antibodies.
  • the idiotope has been shown by immunological and structural techniques to associate partially or entirely with the CDR of a specific mAb (FIG. 2).
  • Idiotopic antibodies are known to have as great or greater affinity toward their specific anti-idiotopic antibody as toward their specific antigen (Braden et al, 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol. 264(1): 137-51).
  • the CDR anti-idiotope adopts a structural conformation of an "internal-image" of the external antigen (Bentley et al, 1990, Three-dimensional structure of an idiotope-anti-idiotope complex, Nature 348(6298): 254-57; Ban et al, 1994, Crystal structure of an idiotope-anti-idiotope Fab complex, Proc. Natl. Acad. Sci. USA 91(5): 1604-08; Poljak, 1994, An idiotope-anti-idiotope complex and the structural basis of molecular mimicking, Proc. Natl. Acad. Sci.
  • idiotopic antibodies are used that have equal or greater affinity towards antigen as anti-idiotopic antibody (Braden et al, 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol. 264(1): 137-51, and references cited therein).
  • antibodies that bind to a peptide of interest and competitively inhibit the binding of the peptide to its receptor can be used to generate anti-idiotope antibodies that "mimic" the peptide receptor and, therefore, bind the peptide.
  • Anti-idiotope antibodies may be generated using techniques well known to those skilled in the art (see, e.g., Greenspan and Bona, 1993, Idiotypes: structure and immunogenicity, FASEB J. 7(5): 437-44; and Nissinoff, 1991, Idiotypes: concepts and applications, J. Immunol. 147(8): 2429-38).
  • Fragment- Anti-idiotopic Antibody E5.2 Fv Fv-Fv idiotope-anti-idiotope complex at Fragment Complex (Mus musculus) 1.9 A resolution, J. Mol Biol. 264(1):
  • Anti-idiotopic Fab 409.5.3 made against Ban et al, 1995, Structure of an an E2 specific feline infectious peritonitis anti-idiotypic Fab against feline peritonitis virus-neutralizing antibody 730.1.4 virus-neutralizing antibody and a comparison with the complexed Fab, FASEB J. 9(1): 107-14
  • specific idiotope/anti-idiotope intermolecular interactions are used as the joining elements to link assembly units together in the staged assembly of a nanostructure (FIG. 1).
  • Each derived assembly unit is designed to contain two specific idiotope/anti-idiotope binding surfaces that are non-cross-reacting. This provides a means of creating a system for the staged assembly of assembly units to form complex nanostructures comprising various and diverse functional elements. Multiple joining pairs can be created by standard methods of phage display (Winter et al, 1994, Making antibodies by phage display technology, Ann. Rev. Immunol. 12: 433-55;Niti et al, 2000, Design and use of phage display libraries for the selection of antibodies and enzymes, Methods Enzymol.
  • one of the CDR domains (i.e., one of the joining elements) of an antibody-derived assembly unit can be engineered as an idiotope.
  • the other CDR can be engineered as a non-complementary anti-idiotope joining element. Since the joining elements are non-identical and non-interactive with each other, this design prevents self-polymerization of the protein component.
  • Such joining elements can be fabricated using combinations of molecular biology and phage display technologies (Winter et al, 1994, Making antibodies by phage display technology, Ann. Rev. Immunol. 12: 433-55; Viti et al, 2000, Design and use of phage display libraries for the selection of antibodies and enzymes, Methods Enzymol. 326: 480-505).
  • the resulting antibody-derived assembly unit will contain both an idiotopic CDR or joining element and a non-complementary anti-idiotopic CDR joining element.
  • the assembly unit to be coupled in the next addition cycle can be designed in an analogous fashion, with a joining element that is an idiotope and a joining element that is a non-complementary anti-idiotope.
  • One CDR of this assembly unit can be engineered to associate with one of the previous CDR components that functions as joining elements. Therefore, in certain embodiments, the CDRs of two adjacent assembly units can be designed to have joining elements that have complementary idiotope/anti-idiotope interactions.
  • assembly units of this design allows for a defined directionality or orientation of the linked assembly unit and of the staged assembly as a whole, i.e., vectorial addition of each assembly unit. Since the CDRs of diabodies are geometrically opposed, the assembly units can be added to an initiator or nanostructure intermediate in known orientation and direction.
  • an assembly unit is fabricated that comprises a diabody unit, wherein the non-complementary joining elements are comprised of two non- complementary idiotopes.
  • a diabody, or a binding derivative or binding fragment thereof may be incorporated into a nanostructure in such a way that only one of the two CDRs is used.
  • the CDRs themselves serve as joining elements, and the body of the diabody between the two CDRs serves as a structural element.
  • Bispecific diabodies are derived from two non-paired scFv fragments.
  • the first portion of the hybrid fragment contains the V H coding region from one F v antibody and the second portion contains the V L coding region derived from another F v antibody.
  • the resulting V H -V L hybrid fragment is joined together by a short Gly 4 Ser linker.
  • the second hybrid fragment will contain linkage of the analogous but opposite coding region pair also joined together by a short Gly 4 Ser linker (FIGS. 2 and 3).
  • the set of hybrid scFv fragments pair by intermolecular interactions between the V H and V L domains.
  • the genes used to create a first assembly unit are derived from the lysozyme idiotopic antibody Dl .3 (represented as V H A and V L A in FIG. 4A) (Braden et al, 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol. 264(1): 137-51) and the feline infectious peritonitis virus-neutralizing idiotopic antibody 730.1.4 (represented as V H B and V L B in FIG. 4A) (Ban et al, 1994, Crystal structure of an idiotype-anti-idiotype Fab complex, Proc. Natl. Acad. Sci.
  • Diabody Unit 1 is represented as A x B in FIG. 4 A.
  • the locations of the promoter (p), ribosome binding site (rbs), pelB leader (pelB), HSV and histidine (his) tags and stop codons (Stop) are also indicated in FIG. 4.
  • the vector system used to engineer the diabody is pET25b (Novagen), which contains a T7 promoter, ribosome binding site, pelB leader sequence, HSV and His tag sequences.
  • FIG. 4B illustrates a second assembly unit (Diabody Unit 2) comprises a diabody, wherein the non-complementary joining elements are designed to contain two non- complementary anti-idiotopes.
  • the genes used to create this second assembly unit are derived from the lysozyme anti-idiotopic antibody E5.2 (represented as V H A' and V L A' in FIG. 4B) (Braden et al, 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol. 264(1): 137-51) and the feline infectious peritonitis virus-neutralizing anti-idiotopic antibody 409.5.3 (represented as V H B' and V L B' in FIG.
  • Diabody Unit 2 is represented as A' x B' .
  • joining elements comprise peptide epitopes.
  • Peptide epitopes may be engineered into assembly units to act as joining elements that form a complementary pair with an antibody or antibody binding fragment, the CDR of which binds to the peptide epitope with specificity.
  • Peptide epitopes can be spliced into multiple defined regions contained within the assembly units described above.
  • Peptides epitopes are particularly preferred as joining elements for use in a number of embodiments, in addition to those embodiments wherein the peptide epitope is used for cross-linking assembly units of adjacent nanostructures together. Therefore, peptide epitopes provide versatility to assembly units into which they are inco ⁇ orated.
  • peptide epitopes can serve as joining elements for junctions that can be initiation points for the assembly of new branches of a nanostructure from a pre-existing branch.
  • Such branching may be used to generate one,- two- or three-dimensional structures. It may be used to expand beyond a simple one-dimensional structure or to attach functional units to a one-dimensional structure.
  • joining elements can serve as the binding sites for the addition of separately-fabricated nanostructure sub-assemblies to nanostructure intermediates, h other embodiments, they can serve as binding sites for antibodies that have linked or bound functional elements.
  • assembly units comprise antibody fragments that comprise peptide epitope joining elements.
  • the inherent flexibility within the Fab fragment may be used advantageously for insertion of a joining element that enables various cross-linked geometries between assembly units of nanostructures in a staged assembly, hi one embodiment, to incorporate the additional intermolecular binding site on the Fab fragment needed for staged assembly, the C-terminal distal end, or the ⁇ -turn regions, are engineered to contain a peptide epitope.
  • Exemplary peptide epitopes are set forth in Table 2.
  • Specific exemplary assembly units are variants of bacteriophage T4 tail fiber protein gp37 in which the C-terminal domain of the polypeptide is modified to include sequences that confer specific binding properties on the entire molecule, e.g., sequences derived from avidin that recognize biotin, sequences derived from immunoglobulin heavy chain that recognize Staphylococcal A protein, sequences derived from the Fab portion of the heavy chain of monoclonal antibodies to which their respective Fab light chain counterparts could attach and form an antigen-binding site, immunoactive sequences that recognize specific antibodies, or sequences that bind specific metal ions. These ligands may be immobilized to facilitate purification and/or assembly.
  • Antibody 8F5 Complexed SEQ ID NO: 1 structure of a human With Peptide From Human rhinovirus neutralizing Rhinovirus (Serotype 2) antibody complexed with a Viral Capsid Protein Vp2 peptide derived from viral (Residues 156 -170) capsid protein VP2, EMBO J. 13(10): 2247-56
  • Antibody binding defines a Epitope structure for an epitope that participates in the PrPC ⁇ >PrPSc conformational change. J. Mol. Biol. 293(4): 855-63
  • a peptide epitope can replace the defined ⁇ -turn motifs contained in the fragment directly.
  • a peptide epitope can be linked to the C-terminal amino acid of the CHI heavy chain (Wallace et al, 2001, Exogenous antigen targeted to FcgammaRI on myeloid cells is presented in association with MHC class I, J. Immunol. Methods 248(1-2): 183-94) by standard methods of molecular biology.
  • Table 3 sets forth examples of identified peptide regions contained in IgG and IgG derivations that are suitable for insertion of joining elements or functional elements.
  • Residue regions are defined in the Fc fragment of the intact IgGl from analysis of the atomic coordinates and numbered according to the residue assignments deposited under entry 1IGY at the Brookhaven National Laboratory protein data bank (BNL-pdb) (Berman et al, 2000, The Protein Data Bank, Nucl. Acids Res. 235-42; 1977, The Protein Data Bank. A computer-based archival file for macromolecular structures, Eur. J. Biochem. 80(2): 319-24).
  • Residue regions are defined in the Fab fragment from analysis of the atomic coordinates and numbered according to the residue assignments deposited under entry ICIC at the BNL-pdb.
  • Residue regions are defined within the scFv fragment from analysis of the atomic coordinates and numbered according to the residue assignments deposited under entry 2AP2 at the BNL-pdb.
  • Residue regions are defined within the diabody fragment from analysis of the atomic coordinates and numbered according to the residue assignments deposited under entry 1LMK at the BNL-pdb.
  • the resulting Fab fragment contains an antigen binding domain, at the N-terminal proximal end of the molecule.
  • the Fab fragment also contains a joining element that is a peptide epitope, inserted at a position in the Fab fragment replacing a defined ⁇ -turn motif, or linked directly to the distal C-terminal end of the Fab fragment.
  • the peptide epitope fused to the Fab fragment serves as a highly specific joining element that can serve as an attachment point, through the recognition and binding of a cognate immunoconjugated functional moiety.
  • Antibodies are multivalent molecules made up of polypeptide chains including light (L) chains of approximately 220 amino acids and heavy (H) chains of 450-575 amino acids. The average molecular weight for an intact IgG molecule is in the range 152-196 kD. Structural studies performed on antibodies have revealed that both the light and heavy chains contain a characteristic domain termed the "immunoglobulin fold.”
  • the immunoglobulin fold is defined as a barrel-shaped sandwich consisting of two layered anti-parallel ⁇ -sheets linked together by a disulfide bond. The predominant secondary structure in an antibody is an anti-parallel ⁇ -sheet with short stretches of ⁇ -helix.
  • the light chains contain two immunoglobulin domains, one at the N-terminal portion, which varies from antibody to antibody V L ), and the other at the C-terminal portion, which is relatively constant (C L ).
  • the heavy chains contain four or five immunoglobulin domains, depending upon the class of immunoglobulin.
  • the N-terminal domain varies (V H ) and the other distal domains remain constant (C H 1, C H 2, C H 3, and, in certain cases C H 4).
  • the units of the light and heavy chains associate through disulfide bonds as well as other non-covalent interactions to form the characteristic Y-shaped dimer composed of two light chains and two heavy chains.
  • the antibody fragment containing the V L chain and the V H chain is termed the F v fragment.
  • Interactions of the variable domains with the constant domains in Fab are not very strong, lending a degree of flexibility and positional variability to the overall structure of the molecule.
  • the N-terminal regions of the two Fab arms bind antigen (Mian et al, 1991, Structure, function and properties of antibody binding sites, J. Mol. Biol. 217(1): 133-51; Wilson et al, 1994, Structure of anti-peptide antibody complexes, Res. Immunol. 145(1): 73-8; Wilson et al, 1994, Antibody-antigen interactions: new structures and new conformational changes, Curr. Opin. Struct. Biol. 4(6): 857-67).
  • the Fab arms are connected by a flexible polypeptide to the third fragment, termed the Fc fragment, which is responsible for triggering effector functions that eliminate the antigen as well as dimerize the antigen binding sites.
  • the Fc portion of the IgG antibody molecule is made up of the two constant domains C H 2 and C H 3.
  • the polypeptide segment connecting the Fab and Fc fragments is defined as the hinge and has variable length and flexibility depending upon the antibody class and isotype. This flexible hinge region provides a natural demarcation between the Fc and Fab fragments of the antibody.
  • the hinge and the Fab elbow or bend contained in an intact IgG molecule allow for significant flexibility between the two antigen binding sites and thus permit numerous cross-linking geometries (FIGS. 5 and 6).
  • the proteins making up native and recombinant antibody fragments are candidates for the structural elements of nanostructures assembled by staged assembly.
  • Antibodies used in the staged assembly methods of the invention include, but are not limited to, IgG monoclonal, humanized or chimeric antibodies.
  • Bmding derivatives or binding fragments of antibodies used in the staged assembly methods of the invention also include, but are not limited to, single chain antibodies (scFv) including monomeric ((scFv) fragments), dimeric ((scFv) 2 or diabodies), trimeric ((scFv) 3 or triabodies) and tetrameric ((scFv) 4 or tetrabodies) single chain antibodies; Fab fragments; F(ab') 2 fragments; and fragments produced by a Fab expression library (Huse et al, 1989, Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda, Science, 246, 1275-81).
  • scFv single chain antibodies
  • Fab fragments F(ab') 2 fragments
  • fragments produced by a Fab expression library Huse et al, 1989, Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda, Science, 246, 1275-8
  • mAbs Monoclonal antibodies
  • mAbs Monoclonal antibodies
  • a mAb to an antigen of interest can be prepared by using any technique known in the art that provides for the production of antibody molecules.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the mAbs that may be used in the methods of the invention may be synthesized by any technique commonly known in the art.
  • human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al, 1983, Construction and testing of mouse— human heteromyelomas for human monoclonal antibody production, Proc. Natl. Acad. Sci. USA.
  • polyclonal antibodies cannot be used as components in the present invention.
  • Polyclonal antibodies represent a population of antibodies in which many molecules of different precise specificity exists. Although they may all bind to a particular antigen, they will bind different parts of the antigen with different geometries, a property that is inconsistent with the precise assembly of a nanostructure.
  • Multispecific antibodies may be used as structural elements for use in the staged assembly methods of the invention.
  • “Specific” or “specificity,” as used herein, refers to the ability of an antibody to bind a defined epitope to one distinct antigen-recognition site. Bispecific antibodies, therefore, comprise two distinct antigen recognition sites, each capable of binding a different antigen. Multispecific antibodies have the ability to bind more than two different epitopes, each through the action of a distinct joining element, i.e., an antigen-recognition site.
  • homogeneous bispecific or multispecific mAbs can be created for use as structural elements, via immortilization of lymphocyte clones, created by fusing myeloma cells with lymphocytes raised against an antigen of interest as described above generally for the production of monoclonal antibodies.
  • multispecific mAbs can be produced in virtually unlimited quantities.
  • multispecific mAbs may be created that specifically target and bind a selected biological substance (see, e.g., Colcher et al, 1999, Single-chain antibodies in pancreatic cancer, Ann. NY Acad. Sci. 880: 263-80; Hudson, 1999, Recombinant antibody constructs in cancer therapy, Curr. Opin.
  • a multispecific mAb for use as a structural element according to the methods of the invention may be a bispecific and/or bivalent mAb.
  • a bispecific antibody has the ability to bind two different epitopes, each contained on a distinct antigen-recognition site.
  • a bivalent antibody has the ability to bind to two different epitopes.
  • Bispecific antibodies may be created using methods well-known in the art (see, e.g., Weiner et al, 1995, Bispecific monoclonal antibody therapy of B-cell malignancy, Leuk. Lymphoma 16(3-4): 199-207; Helfrich et /., 1998, Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas, Int. J.
  • genes encoding antibodies of known specificity may be rescued from hybridoma cell lines and can provide the starting material for cloning the rearranged V L and V H genes thorough employment of recombinant DNA technologies (Ward et al, 1989, Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli, Nature 341(6242): 544-46; Sheets et al, 1998, Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens, Proc. Natl. Acad. Sci. USA 95(11): 6157-62).
  • Universal DNA primers maybe designed to anneal to the target V-domain genes and amplified through employment of the polymerase chain reaction. Through design of restriction sites within these primers, the resulting amplified DNA products can be cloned directly for expression in a range of different hosts including bacteria, yeast, plant and insect cells (Tomlinson et al, 2000, Methods for generating multivalent and bispecific antibody fragments, Methods Enzymol. 326: 461-79). These host cells, rather than hybridoma cell lines, can be used, for the production of recombinant engineered antibodies for use in the methods of the invention.
  • a structural element comprises a diabody fragment.
  • a diabody has two CDRs, and is capable of making two highly specific, non-covalent interactions.
  • a diabody, or a binding derivative or binding fragment thereof may be incorporated into a nanostructure in such a way that only one of the two CDRs is used.
  • the CDRs themselves serve as joining elements, and the body of the diabody between the two CDRs serves as a structural element.
  • FIG. 4 Examples of a structural element comprising a diabody fragment are illustrated in FIG. 4.
  • the diabody expression cassettes represented in FIG. 4 are designed so that the pelB signal sequence spliced to the N-terminus of the V H domains genes coding the diabody fragments are targeted and secreted into the E. coli periplasmic space, where the oxidative environment allows proper folding of the diabody. After induction, the overexpressed diabodies fragments are harvested from the E. coli periplasm according to established protocols well-known in the art.
  • diabodies are engineered to add a hexahistidine tag (His6) at the C-terminus of the V L domains to facilitate purification using an immobilized metal affinity chromatography resin (Scopes, 1994, Protein Purification, Principles and Practice, Third Edition, Springer-Verlag, London, pp. 183-85; Scopes, 1994 Protein Purification: Principles and practice (Springer Advanced texts in Chemistry), Third ed., London). Protein overexpression of diabody assembly unit- 1 (FIG.
  • the number of His 6 tags deteraiines the concentration of imidazole (20-250 mM gradient) at which each protein unit contained in the mixture will elute. Those with no hexahistidine tags will exhibit little or no affinity towards the column resin.
  • Those with one hexahistidine tag will generally elute between 20-40 mM imidazole (bispecific diabody) and those with two hexahistidine tags will generally elute between 50 and 100 mM imidazole. Elution peaks may be detected by UV absorbance and verified with SDS-PAGE, native-PAGE or ELISA assay. Even though the purification procedure described above guards against the isolation of unwanted non-bispecific diabody byproducts, methods are employed to ensure that the isolated diabody of interest has functional bispecificity as disclosed hereinbelow.
  • FIG. 4A depicts an A x B diabody in which the V H and V L domains of A define a lysozyme isotopic antibody (D1.3) and in which the V H and V L domains of B define a virus neutralizing idiotopic antibody (730J.4).
  • the gene encoding V H A and V L B includes a hexahistidine tag, whereas the gene encoding V H B and V L A does not.
  • V H and V L domains of B' define a virus neutralizing idiotopic antibody (409.5.3) and in which the V H and V L domains of A' define a lysozyme isotopic antibody (E5.2).
  • the gene encoding V H B' and V L A' includes a hexahistidine tag, whereas the gene encoding V H A' and V L B' does not.
  • sandwich ELISA or BIAcore protocols may be implemented to determine simultaneous and dual occupancy of both antigen-binding sites (bispecificity), as well as equilibrium constants (Abraham et al, 1996, Determination of binding constants of diabodies directed against prostate-specific antigen using electrochemiluminescence-based immunoassays, J. Mol. Recognit. 9(5-6): 456-61; McGuimiess et al, 1996, Phage diabody repertoires for selection of large numbers of bispecific antibody fragments, Nat. Biotechnol. 14(9): 1149-54; McCall et al, 2001, Increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity, J. Immunol.
  • an idiotype/anti-idiotype binding constant is determined using the BIAcore technique
  • one of the antibodies is dissolved in a liquid phase and the other is coupled to the solid phase.
  • Implementation of this technique permits the determination of the association and dissociation rates (k on and k off respectively) for determination of the dissociation constant (Kd) (Goldbaum et al, 1997, Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype, Proc. Natl. Acad. Sci. USA 94(16): 8697-701).
  • a diabody may comprise one or more sites for the insertion of a joining element, a structural element or a functional element.
  • Table 4 shows peptide regions contained in diabody units that maybe used for the insertion of joining, structural or functional elements.
  • a peptide region is a portion of a protein of interest, e.g., of an antibody or a bmding derivative or binding fragment thereof.
  • a peptide region is preferably exposed on the surface of the protein of interest, and is amenable to being re-engineered through the insertion of additional peptides or the alteration of its sequence or both.
  • Table 4 summarizes the amino acids identified as ⁇ -turns located on the surface of a diabody with V H -V L variable domain linkage (pdb entry 1LMK). Residue regions are defined within the diabody fragment from analysis of the atomic coordinates and numbered according to the residue assignments deposited under entry 1LMK pdb. Chain assignments are labeled in accord with the corresponding deposited pdb coordinates. Table 4: Identified Peptide Regions Contained in Diabody Structural Elements for the Insertion of Joining, Structural or Functional Elements
  • binding sites may be added as joining elements to a diabody to make possible structural branches, forks, T-junctions, or multidimensional architectural binding sites, in addition to the two joining elements formed by the oppositely directed CDRs.
  • Alteration of the sequence of surface loops in proteins appears to have little impact on the overall folding of a protein, and it is frequently possible to make insertion mutants at the sites of ⁇ -turns.
  • the surface loops are the places where sequences can be added to the protein with the lowest probability of disrupting the protein structure.
  • joining elements mays be spliced internal to, or replacing the ⁇ -turn residues as disclosed herein in Table 4. Since the general three-dimensional structure of diabodies is known, and since it is possible to homology-model the three-dimensional structure of diabodies of similar sequence (Guex and Peitsch, 1997, SWISS-MODEL and the Swiss-PdbViewer.
  • a visual investigation of the three-dimensional structure of a diabody is performed with the molecular visualization package QUANTA (Accelrys Inc., San Diego, CA) run on a Silicon Graphics Workstation.
  • the coordinates defining the three-dimensional positions of the atoms of a diabody molecule are included in the PDB entry ILMK.
  • the information provided by the three-dimensional structure of the immunoglobulin being engineered (whether derived directly from X-ray crystallography, or from homology modeling based on a homologous structure) allows the identification of all the amino acids in the protein of interest that correspond to amino acids that constitute surface loops.
  • D ⁇ A encoding a peptide epitope derived from the H-ras protein is inserted into a diabody assembly unit coding sequence at a site defined by visual investigation of the three-dimensional atomic coordinates as determined by x-ray crystallography.
  • the H-ras epitope is flanked by four glycines on either side, to provide flexibility and accessibility for cognate antibody binding.
  • diabody assembly unit/ras peptide protein fusion (represented as B ras x A ) has been expressed and purified, it is characterized for retention of diabody valency and function as well as epitope recognition by the appropriate antibody by methods such as ELISA or BIAcore analysis.
  • a structural element for the staged assembly of a nanostructure comprises an antibody fragment.
  • a fragment includes, but is not limited to, an Fab fragment, or an F(ab') 2 fragment, which can be produced by pepsin digestion of an IgG antibody molecule, thereby releasing the Fc portion. Pepsin digestion can be followed by reducing the disulfide bridges between the resulting F(ab') 2 fragments thereby generating single Fab fragments.
  • Fab fragments are elongated dirigible shaped molecules that contain a monovalent and monospecific CDR at the N-terminal end of the molecule.
  • an assembly unit is engineered from a Fab fragment by inserting a peptide epitope at the C terminal portion of the Fab fragment. Consequently, a peptide fused to the C-terminus of the Fab fragment may act as a target for another engineered Fab, to provide a highly specific and tight interaction between adjacent Fabs in a nanostructure constructed by staged assembly.
  • the size, shape and structure of the Fab fragment (FIG. 6) make it preferred for use as a structural element because it also comprises, by virtue of its structure, a naturally occurring joining element.
  • the flexible elbow bend which is located in the middle of the fragment, allows for alternative geometries (Roux et al, 1997, Flexibility of human IgG subclasses, J. Immunol. 159(7): 3372-82; Roux et al, 1998, Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes: a role for flexibility and geometry, J. Immunol. 161(8): 4083-90).
  • Fab expression libraries may be constructed (Huse et al, 1989, Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda, Science, 246, 1275-81) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • staged assembly of nanostructures can employ, in certain embodiments, structural elements comprising single-chain scFv fragments.
  • An scFv antibody is composed of a fusion peptide that links the carboxyl terminus of the Fv variable heavy chain (V H ) to the amino terminus of the Fv variable light chain (V L ) or vice versa (Freund et al, 1994, Structural and dynamic properties of the Fv fragment and the single-chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional ⁇ MR spectroscopy, Biochemistry 33(11): 3296-303; Hudson et al, 1999, High avidity scFv multimers; diabodies and triabodies, J.
  • Single-chain antibodies may also be used as structural elements for use in the staged assembly methods of the invention.
  • Single-chain antibodies may be produced by the methods of, e.g., Ladner; (U.S. Patent No. 4,946,778, entitled “Single polypeptide chain binding molecules,” issued August 7, 1990); Bird (1988, Single-Chain Antigen-Binding Proteins, Science 242(4877): 423-26); Huston et al. (1988, Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli, Proc. Natl. Acad. Sci. USA 85: 5879-83), or Ward et al, (1989, Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli, Nature 334: 544-46).
  • An scFv fragment is a substructure of a Fab fragment that can be visualized as a Fab fragment, cut in half at the elbow-bend, missing the terminal constant light and heavy chain domains Freund et al, 1994, Structural and dynamic properties of the Fv fragment and the single- chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional NMR spectroscopy, Biochemistry 33(11): 3296-303; Malby et al, 1998, Three-dimensional structures of single-chain Fv-neuraminidase complexes, J. Mol. Biol. 279(4): 901-10) (FIG. 2).
  • scFv are smaller and more globular shaped. While approximately half the size of a Fab fragment, a scFv fragment still contains a functional monovalent/monospecific CDR at the N-terminal portion of the molecule.
  • the scFv represents the minimal antigen binding motif that can be expressed in E. coli.
  • scFv fragments are monovalent, maintaining tertiary and quaternary structures similar to that found in the Fv portion of an intact antibody (FIGS. 5 and 2) (Boulot et al, 1990, Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody Dl .3 and of its complex with the antigen, lysozyme, J. Mol. Biol. 213(4): 617-19; Braden et al, 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol.
  • a Gly/Ser peptide linker that is, optimally, 15 amino acids in length, can be used to join the two variable fragments and help maintain favorable interactions between the V H and V L domains (Perisic et al. 1994, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2(12): 1217-26; Takemura et al, 2000, Construction of a diabody (small recombinant bispecific antibody) using a refolding system, Protein ⁇ ng. 13(8): 583-88; Worn et al, 2001, Stability engineering of antibody single-chain Fv fragments, J. Mol. Biol. 305(5): 989-1010).
  • These Gly/Ser linkers can be used to provide flexibility and protease resistance.
  • scFv antibody fragments have similar function, in terms of antigen recognition and binding, as that of intact antibodies.
  • scFv small cell size
  • CDR complex regional positioning of the CDR
  • One advantage of scFv over Fab fragments is that the technology for engineering and producing scFv's is more advanced (see, e.g., Ward, 1993, Antibody engineering using Escherichia coli as host, Adv. Pharmacol. 24: 1-20; Luo et al, 1996, Construction and expression of bi-functional proteins of single-chain Fv with effector domains, J. Biochem. (Tokyo) 120(2): 229-32; Wu et al, 2000, Designer genes: recombinant antibody fragments for biological imaging, Q.
  • a similar strategy is used to incorporate additional intermolecular binding sites on the scFv as was described above for Fab fragments.
  • the C-terminal distal portion or ⁇ -turn regions can be replaced by defined peptide epitopes such as, but not limited to those provided in Table 2, below. These peptide epitopes can replace defined ⁇ -turn motifs or be directly linked to the C-terminal amino acid of the V H or V L heavy chain (depending upon the order of the linked heavy and light variable domains) (Table 3), by manipulation of the appropriate encoding DNA sequences using recombinant DNA procedures well known in the art.
  • the resulting scFv fragment will contain an antigen binding recognition site on one portion of the scFv fragment and a joining element that is a peptide epitope, either replacing the defined ⁇ -turn motifs, or linked at the C-terminal portion of the scFv fragment.
  • a joining element that is a peptide epitope, either replacing the defined ⁇ -turn motifs, or linked at the C-terminal portion of the scFv fragment.
  • the fused peptide epitope will serve as a highly specific joining element in the formation of a joining pair between adjacent assembly units comprising scFv in a staged assembly.
  • a structural element comprises an antibody fragment such as a bispecific IgG fragment, chimeric IgG fragment or a bispecific heterodimeric F(ab') 2 antibody fragment.
  • IgG molecules such as those created by hybridoma technology, can be produced that are either bivalent or bispecific, using the methods of, e.g., Suresh et al. (1986, Bispecific monoclonal antibodies from hybrid hybridomas, Methods Enzymol. 121 : 210-28); Holliger et al. (1993, Engineering bispecific antibodies, Curr. Opin. Biotechnol.
  • Bispecific IgGs may be created by any method known in the art, e.g., by chemical coupling methodologies or through the development of hybrid hybridoma cell lines (also referred to as hybrid hybridoma technology) (Milstein et al, 1983, Hybrid hybridomas and their use in immunohistochemistry, Nature 305(5934): 537-40) (FIG. 7).
  • bispecific antibodies comprises exposing IgG to limited proteolytic digestion, where the two identical Fab fragments are released from the Fc fragment upon cleavage of the hinge polypeptide (FIG. 8).
  • These single monovalent Fab fragments can be used alone, or chemically linked together (at the hinge cysteines) with a Fab fragment of separate origin to form a bispecific heterodimeric F(ab') 2 .
  • Chemically linked bispecific F(ab') 2 fragments have been studied and evaluated in several small-scale clinical trials (Hudson, 1999, Recombinant antibody constructs in cancer therapy, Curr. Opin. Immunol. 11(5): 548-57; Segal et al, 1999, Bispecific antibodies in cancer therapy, Curr. Opin.
  • bispecific antibodies are produced by replacing the Fc dimer-forming motif with another dimerization motif.
  • leucine zippers that can form heterodimers, such those found in Fos and Jun proteins, are linked to two different Fab portions of an IgG molecule by gene fusion. When expressed individually in an appropriate cell line, the fusion IgG's can be isolated as Fab-(zipper) 2 homodimers. Heterodimer formation is then achieved by reduction of the disulfide bonds within the hinge region of the homodimers to release the monomeric subunits.
  • bispecific heterodimers containing Fos-Jun paired leucine zipper motifs as the majority of the end products.
  • Variations of this technique can be used to produce bispecific Fab and Fv fusion proteins (Kostelny et al, 1992, Formation of a bispecific antibody by the use of leucine zippers, J. Immunol. 148(5): 1547-53; Tso et al, 1995, Preparation of a bispecific F(ab') 2 targeted to the human IL-2 receptor, J. Hematother.
  • Multivalent and multifunctional antibodies of high quality, quantity and purity may be created by recombinant antibody technology ((see , e.g., Morrison et al, 1989, Genetically engineered antibody molecules, Adv. Immunol. 44: 65-92; Shin et al, 1993, Hybrid antibodies, Int. Rev. Immunol. 10(2-3): 177-86; Sensel et al, 1997, Engineering novel antibody molecules, Chem. Immunol. 65: 129-58; Hudson et al, 1998, Recombinant antibody fragments, Curr. Opin. Biotechnol. 9(4): 395-402).
  • human, humanized or chimeric (e.g., human-mouse or human-other species) monoclonal antibodies (mAbs), or binding derivatives or binding fragments thereof, may be used as structural elements for use in the staged assembly methods of the invention.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • Humanized antibodies are also referred to as "chimeric antibodies.”
  • Humanized or chimeric antibodies may be produced by methods well known in the art (see, e.g., Queen, U.S. Patent No. 5,585,089, entitled "Humanized immunoglobulins,” issued December 17, 1996, which is incorporated herein by reference in its entirety).
  • Chimeric antibodies may be used as structural elements according to the methods of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin effector or constant region. Techniques have been developed for the production of chimeric antibodies (Morrison et al, 1984, Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains, Proc. Natl. Acad. Sci.
  • structural elements comprise diabodies or multimeric scFv fragments.
  • scFv fragments especially those with shortened peptide linkers, e.g. 3, 4 or 5 amino acid residues in length, form dimers ((scFv 2 ) or diabodies) rather than monomers in solution (Dolezal et al, 2000, ScFv multimers of the anti-neuraminidase antibody NCI 0: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers, Protein Eng. 13(8): 565-74).
  • Interchain domain interactions rather than intrachain domain interactions, occur in order to form the stable dimeric diabody fragments (Holliger et al, 1993, Diabodies: small bivalent and bispecific antibody fragments, Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-48).
  • a shortened peptide linker may prevent intrachain domain pairing and thus allow formation of interchain interactions that result in diabody fragment formation (Perisic et al 1994, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2(12): 1217-26).
  • diabodies can be used as the structural elements for the staged assembly of one-, two- and three-dimensional nanostructures.
  • the term "diabody” refers to dimeric single-chain variable antibody fragments (scFv).
  • An scFv fragment as described above, is composed of a fusion peptide that links the carboxyl terminus of the Fv variable heavy chain to the amino terminus of the Fv variable light chain (V H -V L ) or vice versa (i.e. V L -V H ) (Pluckthun et al, 1997, New protein engineering approaches to multivalent and bispecific antibody fragments, Immunotechnology 3(2): 83-105; Hudson, 1998, Recombinant antibody fragments, Curr. Opin. Biotechnol. 9(4): 395-402; Kipriyanov et al, 1999, Generation of recombinant antibodies, Mol. BiotechnolJ2(2): 173-201).
  • a diabody or multimeric fragment is thermostable (see, e.g., Jermutus et al, 2001, Tailoring in vitro evolution for protein affinity or stability, Proc. Natl. Acad. Sci. USA 98(1): 75-80; Worn et al, 2001, Stability engineering of antibody single-chain Fv fragments, j. Mol. Biol. 305(5): 989-1010).
  • Thermostability is a useful characteristic for structural elements utilized in the staged assembly of one- two- and three-dimensional nanostructures.
  • a diabody is a bivalent molecule containing "two bodies" that mclude two separate antigen-binding sites in opposition to one another and related by approximately 170° about the pseudo-two-fold axis of symmetry (parallel to the interface)
  • a monospecific diabody contains two identical antigen-binding sites, both with specificity for the same ligand/hapten.
  • a bispecific diabody contains two antigen-binding sites, each specific for a different ligand/hapten; that is, a bispecific diabody is derived from two different non-paired scFv fragments.
  • the first hybrid fragment contains the V H coding region from a first F v antibody and a V L coding region derived from a second F v antibody.
  • the resulting V H -V L hybrid fragment is joined together by a short Gly/Ser linker.
  • the second hybrid fragment contains the V L coding region from the first F v antibody and the V H coding region derived from the second F v antibody.
  • bispecific links permits the creation of bispecific antibody fragments that demonstrate bispecific affinity towards each ligand (Poljak, 1994, An idiotope-anti- idiotope complex and the structural basis of molecular mimicking, Proc. Natl. Acad. Sci. U.S.A. 91(5): 1599-1600; Kipriyanov et al, 1998, Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells, h t. J.
  • Diabodies exhibit several properties that make them particularly attractive for use the in staged assembly methods of the invention: (i) they are structures containing oppositely directed antigen binding sites; (ii) the geometrical opposition of the two antigen-binding sites optimizes the potential for building linear nanostructures or linear extensions of nanostructures; (iii) they have a well-defined size, shape, structure and stoichiometry; (iv) they have structural rigidity and well-defined recognition and binding properties; (v) binding motifs exhibiting specificity for a very broad range of organic and inorganic moieties can be identified and incorporated into a diabody structure (vi) their X-ray structure has been solved (FIG.
  • diabodies form strong intermolecular bonds to one another;
  • the intermolecular bonds are highly specific;
  • the immunoglobulin fold provides a structured protein core (structural element) and a stable spatial relationship among the different faces of the protein;
  • loops in which additional binding sites may be inserted are readily identified through an examination of the three-dimensional structure of a diabody (Zhu et al, 1996, High level secretion of a humanized bispecific diabody from Escherichia coli, Biotechnology (NY) 14(2): 192-96; Hudson et al, 1999, High avidity scFv multimers; diabodies and triabodies, J.
  • scFv fragments can also associate into multivalent multimers (Hudson et al, 1999, High avidity scFv multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89; Power et al, 2000, Synthesis of high avidity antibody fragments (scFv multimers) for cancer imaging, J. Immunol. Methods 242(1-2): 193-204; Todorovska et al, 2001, Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J.
  • Multimer formation is dependent upon the length of the linker used to associate the variable domains (V-domain) together, as well as the V-domain composition and orientation (V H -V L versus V L -V H ). Reducing the linker length below three residues usually favors trimer or triabody formation, e.g., scFv) 3 .
  • Tetrabody formation e.g., (scFv) 4 also has been reported in at least two instances where the linker length was 0 residues in length and the V-domain orientation was V L -V H (Todorovska et al, 2001, Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J. Immunol. Methods 248(1-2): 47-66).
  • An antibody variable domain may functionally comprise both a structural element and a joining element in an assembly unit for staged assembly.
  • the extent of a joining element may not always be completely defined.
  • the ⁇ - sheet structure of an antibody variable domain maintains the geometric relationship between the CDR and the other parts of the molecule. But it is also important for maintaining the structural relationships between the loops of the CDR that provide the binding affinity and specificity of the complementary partner of the joining pair. Consequently, an antibody variable domain may functionally comprise both a structural element and a joining element in an assembly unit.
  • antibody molecules and binding fragments of antibodies are preferred elements of joining elements, they may also provide structural framework for many embodiments, and as described above, for an assembly unit.
  • Antibody assembly units may include antibodies as functional elements. Antibodies, binding fragments and bmding derivatives as described above that are not involved as a joining element or structural element may serve as functional elements for site specific attachment of other moieties. Thus, in certain embodiments, an assembly unit having more than two joining elements is used to build a nanostructure.
  • the additional joining elements can be used, for example: (i) as an attachment point for addition or insertion of a functional element or functional moiety (see Table 4 above); (ii) as the attachment point of the initiator to a solid substrate; or (iii) as attachment points for subassemblies.
  • an assembly unit comprises a structural element.
  • the structural element generally has a rigid structure (although in certain embodiments, described below, the structural element may be non- rigid).
  • the structural element is preferably a defined peptide, protein or protein fragment of known size and structure that comprises at least about 50 amino acids and, generally, fewer than 2000 amino acids. Peptides, proteins and protein fragments are preferred since naturally-occurring peptides, proteins and protein fragments have well-defined structures, with structured cores that provide stable spatial relationships between and among the different faces of the protein. This property allows the structural element to maintain predesigned geometric relationships between the joining elements and functional elements of the assembly unit, and the relative positions and stoichiometries of assembly units to which it is bound.
  • proteins as structural elements has particular advantages over other choices such as inorganic nanoparticles.
  • Most populations of inorganic nanoparticles are heterogeneous, making them unattractive scaffolds for the assembly of a nanostructure.
  • each inorganic nanoparticle is made up of a different number of atoms, with different geometric relationships between facets and crystal faces, as well as defects and impurities.
  • a comparably sized population of proteins is, by contrast, very homogeneous, with each protein comprised of the same number of amino acids, each arranged in approximately the same way, differing in arrangement, for the most part, only through the effect of thermal fluctuations. Consequently, two proteins designed to interact with one another will always interact with the same geometry, resulting in the formation of a complex of predictable geometry and stoichiometry. This property is essential for massively parallel "bottom-up" assembly of nanostructures.
  • a structural element may be used to maintain the geometric relationships among the joining elements and functional elements of a nanostructure.
  • a rigid structural element is generally preferred for construction of nanostructures using the staged assembly methods described herein. This rigidity is typical of many proteins and may be conferred upon the protein through the properties of the secondary structural elements making up the protein, such as -helices and ⁇ -sheets.
  • Structural elements may be based on the structure of proteins, protein fragments or peptides whose three-dimensional structure is known or may be designed ab initio.
  • proteins or protein fragments that may be utilized as structural elements in an assembly unit include, but are not limited to, antibody domains, diabodies, single-chain antibody variable domains, and bacterial pilins.
  • structural elements, joining elements and functional elements may be of well-defined extent, separated, for example, by glycine linkers.
  • joining elements may involve peptides or protein segments that are integral parts of a structural element, or may comprise multiple loops at one end of a structural element, such as in the case of the complementarity determining regions (CDRs) of antibody variable domains (Kabat et al, 1983, Sequences of Proteins of hnmunological hiterest, U.S. Department of Health and Human Services).
  • CDRs complementarity determining regions
  • variable domain represents a structural element and the boundary between the structural element and the CDR making up the joining element (although well-defined in the literature on the basis of the comparisons of many antibody sequences) may not always be completely unambiguous structurally. There may not always be a well-defined boundary between a structural element and a joining element, and the boundary between these domains, although well- defined on the basis of their respective utilities, may be ambiguous spatially.
  • Structural elements of the present invention comprise, e.g., core structural elements of naturally-occurring proteins that are then modified to incorporate joining elements, functional elements, and/or a flexible domain (e.g., a tri-, tetra- or pentaglycine), thereby providing useful assembly units. Consequently, in certain embodiments, structures of existing proteins are analyzed to identify those portions of the protein or part thereof that can be modified without substantially affecting the rigid structure of that protein or protein part.
  • the amino acid sequence of surface loop regions of a protein or structural element are altered with little impact on the overall folding of the protein.
  • the amino acid sequences of a surface loop of a protein are generally preferred as amino acid positions into which the additional amino acid sequence of a joining element, a functional element, and/or a flexible domain maybe inserted, with the lowest probability of disrupting the protein structure.
  • Determining the position of surface loops in a protein is carried out by examination of the three-dimensional structure of the protein or a homolog thereof, if three-dimensional atomic coordinates are available, using, for example, a public-domain protein visualization computer program such as RASMOL (Sayle et al, 1995, RasMol: Biomolecular graphics for all, Trends Biochem. Sci. (TIBS) 20(9): 374-376; Saqi et al, 1994, PdbMotif ⁇ a tool for the automatic identification and display of motifs in protein structures, Comput. Appl. Biosci. 10(5): 545-46). In this manner, amino acids included in surface loops, and the relative spatial locations of these surface loops, can be determined.
  • RASMOL RasMol: Biomolecular graphics for all, Trends Biochem. Sci.
  • the amino acid sequence of the molecule of interest, or a portion thereof can be aligned with that of the molecule whose three-dimensional structure is known.
  • This comparison done, for example, using BLAST (Altschul et al, 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25: 3389-3402) or LALIGN (Huang and Miller, 1991, A time efficient, linear-space local similarity algorithm, Adv. Appl. Math.
  • the invention encompasses structural elements comprising leucine zipper-type coiled coils for use in the construction of nanostructures using the staged assembly methods of the invention.
  • Leucine zippers are well-known, ⁇ - helical protein structures (Oas et al, 1994, Springs and hinges: dynamic coiled coils and discontinuities, TIBS 19: 51-54; Branden et al, 1999, Introduction to Protein Structure 2nd ed., Garland Publishing, Inc., New York) that are involved in the oligomerization of proteins or protein monomers into dimeric, trimeric, and tetrameric structures, depending on the exact sequence of the leucine zipper domain (Harbury et al, 1993, A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants, Science 262: 1401-07).
  • trimeric and tetrameric units may also be used for the construction of assembly units for use in staged assembly of nanostructures according to the methods disclosed herein.
  • trimeric and tetrameric units could be especially useful for incorporation of functional elements that, e.g., require two or more chemical moieties for proper activity, for example, the incorporation of two cysteine moieties for binding of gold particles.
  • Structural elements comprising four-helix bundles, for example linked to antibody- derived domains, can also be employed in assembly units in the nanostructures of the invention.
  • the design and construction of leucine zippers represent one type of a coiled coil oligomerization peptide useful in the construction of a structural element of an assembly unit.
  • Another type is a four-helix bundle, a non-limiting example of which is shown in FIG. 10. Because there are one or more loop segments (i.e. non-helical segments) joining the helices to form an assembly unit, this structure is also called a "helix-loop-helix" structure.
  • the loop sections contribute to the stability of the overall structure by keeping the helices near each other and, therefore, at a functionally high concentration.
  • helix-loop-helix proteins include, but are not limited to: the bacterial Rop protein (a homodimer containing two helix-loop-helix molecules) (Lassalle et al., 1998, Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four alpha-helix bundle protein, J. Mol. Biol.
  • cytochrome b562 a monomeric protein made up of a single helix-loop- helix-loop-helix-loop-helix structure
  • cytochrome b562 a monomeric protein made up of a single helix-loop- helix-loop-helix-loop-helix structure
  • MyoD DNA-binding domain (Ma et al, 1994, Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation, Cell 77(3): 451-59); USF1 and USF2 DNA-binding domains (Ferre-D'Amare et al, 1994, Structure and function of the b/HLH/Z domain of USF, EMBO J. 13(1): 180-9; Kurschner et al, 1997, USF2/FIP associates with the b-Zip transcription factor, c-Maf, via its bHLH domain and inhibits c-Maf DNA binding activity, Biochem. Biophys. Res.
  • Both helical regions and loop regions of the Rop protein exhibit properties that indicate that the Rop protein, or fragments thereof, may be used as structural elements in the construction of assembly units in the staged assembly methods of the invention, h one embodiment, the methods of Munson et al. (1996, What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties, Protein Science 5: 1584-93) are used to mutagenize the a and J residues in the helical regions of the Rop protein to produce variant polypeptides having both increased and decreased thermal stability.
  • functional elements include, but are not limited to, peptides, proteins, protein domains, small molecules, inorganic nanoparticles, atoms, clusters of atoms, magnetic, photonic or electronic nanoparticles.
  • the specific activity or property associated with a particular functional element which will generally be independent of the structural attributes of the assembly unit to which it is attached, can be selected from a very large set of possible functions, including but not limited to, a biological property such as those conferred by proteins (e.g., a transcriptional, translational, binding, modifying or catalyzing property).
  • functional groups may be used that confer chemical, organic, physical electrical, optical, structural, mechanical, computational, magnetic or sensor properties to the assembly unit.
  • functional elements include, but are not limited to: metallic or metal oxide nanoparticles (Argonide Corporation, Sanford, FL; NanoEnergy Corporation, Longmont, CO; Nanophase Technologies Corporation, Romeoville, IL; Nanotechnologies, Austin, TX; TAL Materials, hie, Ann Arbor, MI); gold or gold-coated nanoparticles (Nanoprobes, Inc., Yaphank, NY; Nanospectra LLC, Houston TX); immunoconjugates (Nanoprobes, ie, Yaphank, NY); non-metallic nanoparticles (Nanotechnologies, Austin, TX); ceramic nanofibers (Argonide Corporation, Sanford, FL); fullerenes or nanotubes (e.g., carbon nanotubes) (Materials and Electrochemical Research Corporation, Arlington, AZ; Nanolab, Brighton MA; Nanosys, Inc., Cambridge MA; Carbon Nanotechnologies Incorporated, Houston, TX); nanocrystals (NanoGram Corporation,
  • Functional elements may also comprise any art-known detectable marker, including radioactive labels such as 32 P, 35 S, 3 H, and the like; chromophores; fluorophores; chemiluminescent molecules; or enzymatic markers.
  • a functional element is a fluorophore.
  • exemplary fluorophore moieties that can be selected as labels are set forth in Table 5.
  • DBITC 4-dimethylaminophenylazophenyl-4'-isothiocyanate
  • B-phycoerythrin o-phthaldialdehyde pyrene and derivatives pyrene pyrene butyrate succinimidyl 1 -pyrene butyrate
  • Reactive Red 4 (Cibacron® Brilliant Red 3B-A) rhodamine and derivatives:
  • TAMRA N,N,N',N-tetramethyl-6-carboxyrhodamine
  • TRITC tetramethyl rhodamine tetramethyl rhodamine isothiocyanate
  • a functional element is a chemiluminescent substrate such as luminol (Amersham Biosciences), BOLDTM APB (hitergen), Lumigen APS (Lumigen), etc.
  • the functional element is an enzyme.
  • the enzyme in certain embodiments, may produce a detectable signal when a particular chemical reaction is conducted, such as the enzymes alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, etc.
  • a functional element is a hapten or an antigen (e.g., ras).
  • a functional element is a molecule such as biotin, to which a labeled avidin molecule or streptavidin may be bound, or digoxygenin, to which a labeled anti-digoxygenin antibody may be bound.
  • a functional element is a lectin such as peanut lectin or soybean agglutinin.
  • a functional element is a toxin, such as Pseudomonas exotoxin (Chaudhary et al, 1989, A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin, Nature 339(6223): 394-97).
  • Peptides, proteins or protein domains may be added to proteinaceous assembly units using the tools of molecular biology commonly known in the art to produce fusion proteins in which the functional elements are introduced at the N-terminus of the proteins, the C-terminus of the protein, or in a loop within the protein in such a way as to not disrupt folding of the protein.
  • Non-peptide functional elements may be added to an assembly unit by the incorporation of a peptide or protein moiety that exhibits specificity for said functional element, into the proteinaceous portion of the assembly unit.
  • an entire antibody variable domain e.g. a single-chain variable domain
  • is inco ⁇ orated into an assembly unit e.g. into the joining or structural element thereof, in order to act as an affinity target for a functional element.
  • a flexible segment e.g., a polyglycine peptide sequence
  • This polyglycine linker will act as a flexible spacer that facilitates folding of the original protein after synthesis of the recombinant fusion protein.
  • the antibody domain is chosen for its binding specificity for a functional element, which can be, but is not limited to, a protein or peptide, or to an inorganic material.
  • a functional element may be a quantum dot (semiconductor nanocrystal, e.g., QDOTTM, Quantum Dot Corporation, Hayward, CA) with desirable optical properties.
  • a quantum dot can be incorporated into a nanostructure through a peptide that has specificity for a particular class of quantum dot.
  • identification of such a peptide, having a required affinity for a particular type of quantum dot is carried out using methods well known in the art. For example, such a peptide is selected from a large library of phage-displayed peptides using an affinity purification method.
  • Suitable purification methods include, e.g., biopanning (Whaley et al, 2000, Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly, Nature 405(6787): 665-68) and affinity column chromatography.
  • biopanning Wang et al, 2000, Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly, Nature 405(6787): 665-68
  • affinity column chromatography affinity column chromatography.
  • affinity purification is an iterative process that uses several affinity purification steps.
  • Affinity purification may been used to identify peptides with affinity for particular metals and semiconductors (Belcher, 2001, Evolving Biomolecular Control of Semiconductor and Magnetic Nanostructure, presentation at Nanoscience: Underlying Physical Concepts and Properties, National Academy of Sciences, Washington, D.C., May 18-20, 2001; Belcher et al, 2001, Abstracts of Papers, 222nd ACS National Meeting, Chicago, IL, United States, August 26-30, 2001, American Chemical Society, Washington, D.C.).
  • a functional element is incorporated into a nanostructure through the use of joining elements (interaction sites) by which non-proteinaceous nanoparticles having desirable properties are attached to the nanostructure.
  • joining elements are, in two non-limiting examples, derived from the complementarity determining regions of antibody variable domains or from affinity selected peptides.
  • the unique, tunable properties of semiconductor nanocrystals make them preferable for use in nanodevices, including photoconductive nanodevices and light emitting diodes.
  • the electrical properties of an individual nanostructure are difficult to measure, and therefore, photoconductivity is used as a measure of the properties of those nanostructures.
  • Photoconductivity is a well-known phenomena used for analysis of the properties of semiconductors and organic solids. Photoconductivity has long been used to transport electrons between weakly interacting molecules in otherwise insulating organic solids.
  • Photocurrent spectral responses may also be used to map the absorption spectra of the nanocrystals in nanostructures and compared to the photocurrent spectral responses of individual nanocrystals (see, e.g., Murray et al, 2000, Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies, Ann. Rev. Material Science 30: 545-610).
  • optical and photoluminescence spectra may also be used to study the optical properties of nanostructures (see, e.g., Murray et al, 2000, Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies, Ann. Rev. Material Science 30: 545-610).
  • biosensors are commercially available that are made of a combination of proteins and quantum dots (Alivisatos et al, 1996, Organization of 'nanocrystal molecules' using DNA, Nature 382: 609-11; Weiss et al, U.S. Patent No. 6,207,392 entitled "Semiconductor nanocrystal probes for biological applications and process for making and using such probes," issued March 27, 2001).
  • the ability to complex a quantum dot with a highly specific biological molecule e.g., a single stranded DNA or an antibody molecule
  • Using different sized quantum dots each complexed to a molecule with different specificity, allows multiple sensing of components simultaneously.
  • Inorganic structures such as quantum dots and nanocrystals of metals or semiconductors may be used in the staged assembly of nanostructures as termini of branches of the assembled nanostructure. Once such inorganic structures are added, additional groups cannot be attached to them because they have an indeterminate stoichiometry for any set of binding sites engineered into a nanostructure. This influences the sequence in which assembly units are added to form a nanostructure being fabricated by staged assembly. For example, once a particular nanocrystal is added to the nanostructure, it is generally not preferred to add additional assembly units with joining elements that recognize and bind that type of nanocrystal, because it is generally not possible to control the positioning of such assembly units relative to the nanocrystal.
  • a rigid nanostructure fabricated according to the staged assembly methods of the present invention, comprises a magnetic nanoparticle attached as a functional element to the end of a nanostructure lever arm, which acts as a very sensitive sensor of local magnetic fields.
  • the presence of a magnetic field acts to change the position of the magnetic nanoparticle, bending the nanostructure lever arm relative to the solid substrate to which it is attached.
  • the position of the lever arm may be sensed, in certain embodiments, through a change in position of, for example, optical nanoparticles attached as functional elements to other positions (assembly units) along the nanostructure lever arm.
  • the degree of movement of the lever arm is calibrated to provide a measure of the magnetic field.
  • nanostructures that are fabricated according to the staged assembly methods of the invention have desirable properties in the absence of specialized functional elements, h such embodiments, a staged assembly process provides a two-dimensional or a three-dimensional nanostructure with small (nanometer-scale), precisely-sized, and well-defined pores that can be used, for example, for filtering particles in a microfluidic system.
  • nanostructures are assembled that not only comprise such well-defined pores but also comprise functional elements that enhance the separation properties of the nanostructure, allowing separations based not only on size but also with respect to the charge and/or hydrophilicity or hydrophobicity properties of the molecules to be separated.
  • Such nanostructures can be used for HPLC separations, providing extremely uniform packing materials and separations based upon those materials.
  • functional elements include, but are not limited to, peptide sequences comprising one or more side chains that are positively or negatively charged at a pH used for the desired chromatographic separation; and peptide sequences comprising one or more amino acids having hydrophobic or lipophilic side chains.
  • junctions are architectural structures that can serve as "switch points" in microelectronic circuits such as silicon based electronic chips, etc.
  • multivalent antibodies or bindmg derivatives or binding fragments thereof are used as junction structures and are introduced into nanostructures using the methods of the present invention.
  • bioelectronic and biocomputational devices comprising these nanostructure junctions are quantum cellular automata (QCA).
  • a molecular clone of an antibody to an antigen of interest can be prepared by techniques well-known in the art. Recombinant DNA methodology may be used to construct nucleic acid sequences that encode a monoclonal antibody molecule, or antigen binding region thereof (see, e.g., Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Chapters 1, 2, 3, 5, 6, 8, 9, 10, 13, 14, 15 and 18, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel et al, 1989, Current Protocols in Molecular Biology, Chapters 1, 2, 3, 5, 6, 8, 10, 11, 12, 15, 16, 19, 20 and 24, Green Publishing Associates and Wiley hiterscience, N.Y.; Current Protocols in Immiinology, Chapters 2, 8, 9, 10, 17 and 18, John Wiley & Sons, 2001, Editors John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, Series Editor: Richard Coico).
  • Antibodies can be expressed in bacteria either intracellularly or extracellularly by secretion into the bacterial periplasm (Tomlinson and Holliger, 2000, Methods for generating multivalent and bispecific antibody fragments, Methods Enzymol. 326: 461-79). Intracellular expression of recombinant antibodies, however, frequently leads to the formation of insoluble aggregates of the protein, which are referred to as inclusion bodies, presumably due to the non-reducing environment of the bacterial cytoplasm, which inhibits disulfide bond formation between antibody domains. It is possible to refold the antibodies into functional proteins through solubilization of the inclusion bodies with strong denaturants followed by exposure to renaturing conditions, by methods commonly known in the art.
  • a coding sequence for a bacterially-derived periplasmic signal sequence can be spliced at the N-terminal portion of the gene encoding the antibody to direct the recombinant protein to the bacterial periplasm.
  • the oxidizing environment of the periplasmic space favors proper folding of the antibody domains, including disulfide bond formation.
  • the success of these methods in producing good yields of functional antibody can depend upon the antibody type, derivation and method of overproduction (see Ward, 1992, Antibody engineering: the use of Escherichia coli as an expression host, FASEB J. 6(7): 2422-27; Ward, 1993, Antibody engineering using Escherichia coli as host, Adv. Pharmacol.
  • Antibody molecules may be purified by techniques well-known in the art, e.g., immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.
  • Antibody assembly units may be assembled to form nanostructures by staged assembly.
  • Staged assembly enables massively parallel synthesis of complex, non-periodic, multi-dimensional nanostructures in which organic and inorganic moieties are placed, accurately and precisely, into a pre-designed, three-dimensional architecture.
  • a staged assembly a series of assembly units is added in a given pre-designed order to an initiator unit and/or nanostructure intermediate. Because a large number of identical initiators are used and because subunits are added to all initiators/intermediates simultaneously, staged assembly fabricates multiple identical nanostructures in a massively parallel manner.
  • the initiator units are bound to a solid substrate, support or matrix.
  • Additional assembly units are added sequentially in a procedure akin to solid phase polymer synthesis.
  • the intermediate stage(s) of the nanostructure while it is being assembled, and which comprises the bound assembly units formed on the initiator unit, is generally described as either a nanostructure intermediate or simply, a nanostructure. Addition of each assembly unit to the nanostructure intermediate undergoing assembly depends upon the nature of the joining element presented by the previously added assembly unit and is independent of subsequently added assembly units. Thus assembly units can bind only to the joining elements exposed on the nanostructure intermediate undergoing assembly; that is, the added assembly units do not self-interact and/or polymerize.
  • unbound assembly units do not form dimers or polymers.
  • An assembly unit to be added is preferably provided in molar excess over the initiator unit or nanostructure intermediate in order to drive its reaction with the intermediate to completion. Removal of unbound assembly units during staged assembly is facilitated by carrying out staged assembly using a solid-substrate-bound initiator so that unbound assembly units can be washed away in each cycle of the assembly process.
  • This scheme provides for assembly of complex nanostructures using relatively few non-cross-reacting, complementary joining pairs. Only a few joining pairs need to be used, since only a limited number of joining elements will be exposed on the surface of an assembly intermediate at any one step in the assembly process. Assembly units with complementary joining elements can be added and incubated against the nanostructure intermediate, causing the added assembly units to be attached to the nanostructure intermediate during an assembly cycle. Excess assembly units can then be washed away to prevent them from forming unwanted interactions with other assembly units during subsequent steps of the assembly process. Each position in the nanostructure can be uniquely defined through the process of staged assembly and distinct functional elements can be added at any desired position.
  • the staged assembly method of the invention enables massive parallel manufacture of complex nanostructures, and different complex nanostructures can be further self-assembled into higher order architectures in a hierarchic manner.
  • FIG. 11 depicts an embodiment of the staged assembly method of the invention in one dimension.
  • an initiator unit is immobilized on a solid substrate
  • an assembly unit is added to the initiator (i.e. the matrix bound initiator unit), resulting in a nanostructure intermediate composed of two units. Only a single assembly unit is added in this step, because the second assembly unit cannot interact (i.e. polymerize) with itself.
  • the initiator unit may contain an added functional element and/or may comprise a structural unit of different length from previously added units.
  • a third assembly unit is added that comprises a functional element.
  • additional assembly units are added, each with a designed functional group.
  • the third, fourth and fifth assembly units each carry a unique functional element (designated by geometric shapes protruding from the top of the assembly units in the figure).
  • staged assembly depicted in FIG. 11 requires only two non-cross-reacting, complementary joining pairs.
  • Self-assembly of the structure, as it stands at the end of step 5, would require four non-cross-reacting, complementary joining pairs.
  • This relatively modest improvement in number of required joining pairs becomes far greater as the size of the structure increases. For instance, for a linear structure of N units assembled by an extension of the five steps illustrated in FIG. 11, staged assembly would still require only two non-cross-reacting, complementary joining pairs, whereas self-assembly would require (N-l) non-cross-reacting, complementary joining pairs.
  • the number of nanostructures fabricated is determined by the number of initiator units bound to the matrix while the length of each one-dimensional nanostructure is a function of the number of assembly cycles performed. If assembly units with one or more different functional elements are used, then the order of assembly will define the relative spatial orientation of each functional element relative to the other functional elements.
  • excess unbound assembly units are removed from the attached nanostructure intermediate by a removal step, e.g., a washing step.
  • the substrate-bound nanostructure intermediate may be washed with an appropriate solvent (e.g., an aqueous solution or buffer).
  • the solvent must be able to remove subunits held by non-specific interactions without disrupting the specific, interactions of complementary joining elements.
  • Appropriate solvents may vary as to pH, salt concentration, chemical composition, etc., as required by the assembly units being used.
  • a buffer used for washing the nanostructure intermediate can be, for example, a buffer used in the wash steps implemented in ELISA protocols, such as those described in Current Protocols in Immunology (see Chapter 2, Antibody Detection and Preparation, Section 2J "Enzyme-Linked Immunosorbent Assays," John Wiley & Sons, 2001, Editors John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, Series Editor: Richard Coico).
  • an assembled nanostructure is "capped” by addition of a “capping unit,” which is an assembly unit that carries only a single joining element.
  • the nanostructure can be removed from the solid substrate and isolated.
  • the completed nanodevice will be functional while attached to the solid substrate and need not be removed.
  • step of adding assembly units can be repeated in an iterative manner until a complete nanostructure is assembled, after which time the complete nanostructure can be released by breaking the bond immobilizing the first assembly unit from the matrix at a designed releasing moiety (e.g. a protease site) within the initiator unit or by using a pre-designed process for release (e.g., lowering of pH).
  • a designed releasing moiety e.g. a protease site
  • a pre-designed process for release e.g., lowering of pH.
  • the process of staged assembly as illustrated in FIGS. 11 and 12 is one of the simplest embodiments contemplated for staged assembly.
  • assembly units with additional joining elements can be used to create more complex assemblies. Assembly units may be added individually or, in certain embodiments, they can be added as subassemblies (FIG. 12).
  • compositions and methods disclosed herein provide means for the assembly of these complex, designed nanostructures and of more complex nanodevices formed by the staged assembly of one or a plurality of nanostructures into a larger structure.
  • Fabrication of multidimensional nanostructures can be accomplished, e.g., by incorporating precisely-spaced assembly units containing additional joining elements into individual, one-dimensional nanostructures, where those additional joining elements can be recognized and bound by a suitable cross-linking agent to attach the individual nanostructures together.
  • cross-linking could be, e.g., an antibody or a binding derivative or a binding fragment thereof.
  • the initiator unit is tethered to a solid support.
  • Such tethering is not random (i.e., is not non-specific binding of protein to plastic or random biotinylation of an assembly unit followed by binding to immobilized streptavidin) but involves the binding of a specific element of the initiator unit to the matrix or substrate.
  • the staged assembly process is a vectorial process that requires an unobstructed joining element on the initiator unit for attachment of the next assembly unit. Random binding of initiator units to substrate would, in some cases, result in the obstruction of the joining element needed for the attachment of the next assembly unit, and thus lowering the number of initiator units on which nanostructures are assembled.
  • the initiator unit is not immobilized to a solid substrate.
  • a removal step e.g., a washing step
  • a destructive treatment e.g., protease treatment or chemical degradation
  • Proteins have well-defined binding properties, and the technology to manipulate the intermolecular interactions of proteins is well known in the art (Hayashi et al, 1995, A single expression system for the display, purification and conjugation of single-chain antibodies, Gene 160(1): 129-30; Hayden et al, 1997, Antibody engineering, Curr. Opin. Immunol. 9(2): 201-12; Jung et al, 1999, Selection for improved protein stability by phage display, J. Mol. Biol. 294(1): 163-80, Viti et al, 2000, Design and use of phage display libraries for the selection of antibodies and enzymes, Methods Enzymol.
  • staged assembly of nanostructures need not be limited to components composed primarily of biological molecules, e.g., proteins and nucleic acids, that have specific recognition properties.
  • biological molecules e.g., proteins and nucleic acids
  • optical, magnetic or electrical properties of inorganic atoms or molecules will be required for some embodiments of nanostructures fabricated by staged assembly.
  • inorganic nanoparticles are added to components that are assembled into nanostructures using the staged assembly methods of the invention. This may be done using joining elements specifically selected for binding to inorganic particles. For example, Whaley and co-workers have identified peptides that bind specifically to semiconductor binding surfaces (Whaley et al, 2000, Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly, Nature 405: 665-68). In one embodiment, these peptides are inserted into protein components described herein using standard cloning techniques. Staged assembly of protein constructs as disclosed herein, provides a means of distributing these binding sites in a rigid, well-defined three- dimensional array.
  • the inorganic nanoparticles can be added using a cycle of staged assembly analogous to that used to add proteinaceous assembly units. To accomplish this, it may be necessary, in certain embodiments to adjust the solution conditions under which the nanostructure intermediates are incubated, in order to provide for the solubility of the inorganic nanoparticles. Once an inorganic nanoparticle is added to the nanostructure intermediate, it is not possible to add further units to the inorganic nanoparticle in a controlled fashion because of the microheterogeneities intrinsic to any population of inorganic nanoparticles. These heterogeneities would render the geometry and stoichiometry of further interactions uncontrollable.
  • FIG. 13 is a diagram illustrating the addition of protein units and inorganic elements to a nanostructure according to the staged assembly methods of the invention, hi step 1, an initiator unit is bound to a solid substrate. In step 2, an assembly unit is bound specifically to the initiator unit. In step 3, an additional assembly unit is bound to the nanostructure undergoing assembly.
  • This assembly unit comprises an engineered binding site specific for a particular inorganic element, hi step 4, the inorganic element (depicted as a cross-hatched oval) is added to the structure and bound by the engineered binding site.
  • Step 5 adds another assembly unit with a binding site engineered for specificity to a second type of inorganic element, and that second inorganic element (depicted as a hatched diamond) is added in step 6.
  • the order in which assembly units are added is determined by the desired structure and/or activity that the product nanostructure, and the need to minimize the number of cross-reacting joining element pairs used in the assembly process. Hence determining the order of assembly is an integral part of the design of a nanostructure to be fabricated by staged assembly.
  • Joining elements are chosen, by design, to permit staged assembly of the desired nanostructure. Since the choice of joining element(s) is generally independent of the functional elements to be incorporated into the nanostructure, the joining elements are mixed and matched as needed to fabricate assembly units with the necessary functional elements and joining elements that will provide for the placement of those functional elements in the desired spatial orientation.
  • assembly units comprising two joining elements, designed using the six joining elements that make up three joining pairs, can include any of 18 pairs of the joining elements that are non-interacting. There are 21 possible pairs of joining elements, but three of these pairs are interacting (e.g. A- A') and their use in an assembly unit would lead to the self-association of identical assembly units with one another.
  • joining elements are denoted as A, A', B, B', C and C, where A and A', B and B', and C and C are complementary pairs of joining elements (joining pairs), i.e. they bind to each other with specificity, but not to any of the other four joining elements depicted.
  • each assembly unit further comprises a unique functional element, one of a set of six, and represented as F j to F 6 . According to these conventions, six possible assembly units can be designated as:
  • A-F.-C Staged assembly according to the methods disclosed herein can be used to assemble the following illustrative linear, one-dimensional nanostructures, in which the order and relative vectorial orientation of each assembly unit is independent of the order of the functional elements (the symbol •- is used to represent the solid substrate to which the initiator is attached and a double colon represents the specific interaction between assembly units):
  • a large number of unique assembly units can be constructed using a small number of complementary joining elements. Moreover, only a small number of complementary joining elements are required for the fabrication of a large number of unique and complex nanostructures, since only one type of assembly unit is added in each staged assembly cycle and, therefore, joining elements can be used repeatedly without rendering ambiguous the position of an assembly unit within the completed nanostructure.
  • Attachment of each assembly unit to an initiator or nanostructure intermediate is mediated by formation of a specific joining-pair interaction between one joining element of the assembly unit and one or more unbound complementary joining elements carried by the initiator or nanostructure intermediate.
  • only a single unbound complementary joining element will be present on the initiator or nanostructure intermediate.
  • the staged assembly proceeds by the parallel addition of assembly units, but only a single unit will be attached at any one site on the intermediate, and assembly at all sites that are involved will occur in a pre-designed, vectorial manner.
  • Structural integrity of the nanostructure is of critical importance throughout the process of staged assembly, and the assembly units are preferably connected by noncovalent interactions.
  • a specific non-covalent interaction is, for example, an interaction that occurs between an assembly unit and a nanostructure intermediate.
  • the specific interaction should exhibit adequate affinity to confer stability to the complex between the assembly unit and the nanostructure intermediate sufficient to maintain the interaction stably throughout the entire staged assembly process.
  • a specific non-covalent interaction should exhibit adequate specificity such that the added assembly unit will form stable interactions only with joining elements designed to interact with it.
  • the interactions that occur among elements during the staged assembly process disclosed herein are preferably operationally "irreversible.”
  • a binding constant that meets this requirement cannot be defined unambiguously since "irreversible” is a kinetic concept, and a binding constant is based on equilibrium properties. Nevertheless, interactions with Kd's of the order of 10 "7 or lower (i.e. higher affinity and similar to the Kd of a typical diabody-epitope complex) will typically act "irreversibly" on the time scale of interest, i.e. during staged assembly of a nanostructure.
  • nanostructures fabricated according to the staged assembly methods disclosed herein are subsequently stabilized by chemical fixation (e.g., by fixation with paraformaldehyde or glutaraldehyde) or by cross-linking.
  • the fabrication of a nanostructure by the staged assembly methods of the present invention involves joining relatively rigid and stable assembly units, using non-covalent interactions between and among assembly units. Nevertheless, the joining elements that are incorporated into useful assembly units can be rather disordered, that is, neither stable nor rigid, prior to interaction with a second joining element to form a stable, preferably rigid, joining pair. Therefore, in certain embodiments of the invention, individual assembly units may include unstable, flexible domains prior to assembly, which, after assembly, will be more rigid. In preferred embodiments, a nanostructure fabricated using the compositions and methods disclosed herein is a rigid structure.
  • analysis of the rigidity of a nanostructure is carried out using methods well-known in the art, such as electron microscopy.
  • structural rigidity can be tested by attaching one end of a completed nanostructure directly to a solid surface, i.e., without the use of a flexible tether.
  • the other end of the nanostructure (or a terminal branch of the nanostructure, if it is a multi-branched structure) is then attached to an atomic force microscope (AFM) tip, which is movable. Force is applied to the tip in an attempt to move it.
  • AFM atomic force microscope
  • Force is applied to the tip in an attempt to move it.
  • the nanostructure is flexible, there will be an approximately proportional relationship between the force applied and tip movement as allowed by deflection of the nanostructure.
  • the nanostructure is rigid, there will be little or no deflection of the nanostructure and tip movement as the level of applied force increases, up until the point at which the rigid nanostructure breaks. At that point, there will be a large movement of the AFM tip even though no further force is applied.
  • this method will provide a useful measurement of rigidity.
  • each position in a nanostructure is distinguishable from all others, since each assembly unit can be designed to interact tightly, specifically, and uniquely with its neighbors.
  • Each assembly unit can have an activity and/or characteristic that is distinct to its position within the nanostructure.
  • Each position in the nanostructure is uniquely defined through the process of staged assembly, and through the properties of each assembly unit and/or functional element that is added at a desired position.
  • the staged-assembly methods and assembly units disclosed herein are amenable to large scale, massively parallel, automated manufacturing processes for construction of complex nanostructures of well-defined size, shape, and function.
  • compositions of the present invention capitalize upon the precise dimensions, uniformity and diversity of spatial geometries that proteins are capable of that are used in the construction of the assembly units employed herein. Furthermore, as described hereinbelow, the methods of the mvention are advantageous because genetic engineering techniques can be used to modify and tailor the properties of those biological materials used in the methods of the invention disclosed herein, as well as to synthesize large quantities of such materials in microorganisms.
  • An initiator assembly unit is the first assembly unit incorporated into a nanostructure that is formed by the staged assembly method of the invention.
  • An initiator assembly unit may be attached, in certain embodiments, by covalent or non-covalent interactions, to a solid substrate or other matrix.
  • An initiator assembly unit is also known as an "initiator unit.”
  • Staged assembly of a nanostructure begins by the non-covalent, vectorial addition of a selected assembly unit to the initiator unit.
  • an assembly unit is added to the initiator unit through (i) the incubation of an initiator unit, which in some embodiments, is immobilized to a matrix or substrate, in a solution comprising the next assembly unit to be added.
  • This incubation step is followed by (ii) a removal step, e.g., a washing step, in which excess assembly units are removed from the proximity of the initiator unit.
  • Assembly units bind to the initiator unit through the formation of specific, noncovalent bonds.
  • the joining elements of the next assembly unit are chosen so that they attach only at pre-designated sites on the initiator unit. Only one assembly unit can be added to a target joining element on the initiator unit during the first staged-assembly cycle, and binding of the assembly unit to the target initiator unit is vectorial.
  • Staged assembly continues by repeating steps (i) and (ii) until all of the desired assembly units are incorporated into the nanostructure according to the desired design of the nanostructure.
  • an initiator unit is immobilized on a substrate and additional units are added sequentially in a procedure analogous to solid phase polymer synthesis.
  • An initiator unit is a category of assembly unit, and therefore can comprise any of the structural, joining, and/or functional elements described hereinbelow as being comprised in an assembly unit of the invention.
  • An initiator unit can therefore comprise any of the following molecules, or a binding derivative or binding fragment thereof: a monoclonal antibody; a multispecific antibody, a Fab or F(ab') 2 fragment, a single-chain antibody fragment (scFv); a bispecific, chimeric or bispecific heterodimeric F(ab') 2 ; a diabody or multimeric scFv fragment; a bacterial pilin protein, a leucine zipper-type coiled coil, a four-helix bundle, a peptide epitope, or a PNA, or any other type of assembly unit disclosed herein.
  • the invention provides an initiator assembly unit which comprises at least one joining element, hi other embodiments, the invention provides an initiator assembly unit with two or more joining elements.
  • Initiator units may be tethered to a matrix in a variety of ways.
  • the choice of tethering method will be determined by several design factors including, but not limited to: the type of initiator unit, whether the finished nanostructure must be removed from the matrix, the chemistry of the finished nanostructure, etc.
  • Potential tethering methods include, but are not limited to, antibody binding to initiator epitopes, His tagged initiators, initiator units containing matrix binding domains (e.g., chitin-binding domain, cellulose- binding domain), antibody binding proteins (e.g., protein A or protein G) for antibody or antibody-derived initiator units, streptavidin binding of biotinylated initiators, PNA tethers, and specific covalent attacliment of initiators to matrix.
  • matrix binding domains e.g., chitin-binding domain, cellulose- binding domain
  • antibody binding proteins e.g., protein A or protein G
  • streptavidin binding of biotinylated initiators e.g., PNA tethers, and specific covalent attacliment of initiators to matrix.
  • an initiator unit is immobilized on a solid substrate.
  • Initiator units may be immobilized on solid substrates using methods commonly used in the art for immobilization of antibodies or antigens. There are numerous methods well known in the art for immobilization of antibodies or antigens. These methods include non-specific adsorption onto plastic ELISA plates; biotinylation of a protein, followed by immobilization by binding onto streptavidin or avidin that has been previously adsorbed to a plastic substrate (see, e.g., Sparks et al, 1996, Screening phage-displayed random peptide libraries, in Phage Display of Peptides and Proteins, A Laboratory manual, editors, B.K. Kay, J. Winter and J.
  • protein may be immobilized onto any number of other solid supports such as Sepharose (Dedman et al, 1993, Selection of target biological modifiers from a bacteriophage library of random peptides: the identification of novel calmodulin regulatory peptides, J. Biol. Chem. 268; 23025-30) or paramagnetic beads (Sparks et al, 1996, Screening phage-displayed random peptide libraries, in Phage Display of Peptides and Proteins, A Laboratory manual, editors, B.K. Kay, J. Winter and J. McCafferty, Academic Press, San Diego, pp.
  • Sepharose Dedman et al, 1993, Selection of target biological modifiers from a bacteriophage library of random peptides: the identification of novel calmodulin regulatory peptides, J. Biol. Chem. 268; 23025-30
  • paramagnetic beads Sparks et al, 1996, Screening phage-displayed random peptide libraries
  • Additional methods that may be used include immobilization by reductive amination of amine-containing biological molecules onto aldehyde-containing solid supports (Hermanson, 1996, Bioconjugate Techniques, Academic Press, San Diego, p. 186), and the use of dimethyl pimelimidate (DMP), a homobifunctional cross-linking agent that has imidoester groups on either end (Hermanson, 1996, Bioconjugate Techniques, Academic Press, San Diego, pp. 205-06).
  • DMP dimethyl pimelimidate
  • This reagent has found use in the immobilization of antibody molecules to insoluble supports containing bound protein A (e.g., Schneider et al, 1982, A one-step purification of membrane proteins using a high efficiency immunomatrix, J. Biol. Chem. 257, 10766-69).
  • an initiator unit is a diabody that comprises a tethering domain (T) that recognizes and binds an immobilized antigen/hapten and an opposing domain (A) to which additional assembly units are sequentially added in a staged assembly.
  • Antibody 8F5 which is directed against the antigenic peptide VKAETRLNPDLQPTE (SEQ ID NO: 1) derived human rhinovirus (Serotype 2) viral capsid protein Vp2, is used as the T domain (Tormo et al, 1994, Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2, EMBO J. 13(10): 2247-56).
  • the A domain is the same lysozyme anti- idiotopic antibody (E5.2) previously described for Diabody Unit 1.
  • the completed initiator assembly unit therefore contains 8F5 x 730.1.4 (T x A ) as the opposing CDRs.
  • the initiator unit is constructed and functionally characterized using the methods described herein for characterizing joining elements and/or structural elements comprising diabodies.
  • the rhinovirus antigenic peptide may fused to the protease recognition peptide factor Xa through a short flexible linker spliced at the N termini of the Factor Xa sequence, IEGR, (Nagai and Thogersen, 1984, Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli, Nature309(5971): 810-12) and between the Factor Xa sequence and the antigenic peptide sequence.
  • IEGR short flexible linker spliced at the N termini of the Factor Xa sequence
  • This fusion peptide may be covalently linked to CH-Sepharose 4B (Pharmacia); a sepharose derivative that has a six-carbon long spacer arm and permits coupling via primary amines. (Alternatively, Sepharose derivatives for covalent attachment via carboxyl groups may be used.)
  • the covalently attached fusion protein will serve as a recognition epitope for the tethering domain "8F5" in the initiator unit (T x A ).
  • diabody assembly units 1 and 2 may be sequentially added in a staged assembly, unidirectionally from binding domain A'.
  • the nanostructure may be either cross-linked to the support matrix or released from the matrix upon addition of the protease Factor Xa.
  • the protease will cleave the covalently attached antigenic /Factor Xa fusion peptide, releasing the intact nanostructure from the support matrix, since, by design, there are no Factor Xa recognition sites contained within any of the designed protein assembly units.
  • joining elements suitable for use in the methods of the invention are screened and their interactions identified using antibody- phage-display technology.
  • Phage-display technology for production of recombinant antibodies, or binding derivatives or binding fragments thereof can be used to produce proteins capable of binding to a broad range of diverse antigens, both organic and inorganic (e.g. proteins, peptides, nucleic acids, sugars, and semiconducting surfaces, etc.).
  • Methods for phage-display technology are well known in the art (see, e.g., Marks et al, 1991, By-passing immunization: human antibodies from V-gene libraries displayed on phage, J. Mol. Biol.
  • antibody-phage-display technology is used to overcome these limitations, so that mAbs that recognize particular antigens of interest can be generated more effectively (for methods, see Winter et al, 1994, Making antibodies by phage display technology, Ann. Rev. Immunol.
  • the Fv or Fab antigen-binding portions of V L and the V H genes are "rescued” by PCR amplification using the appropriate primers, from cDNA derived from human spleen or human peripheral blood lymphocyte cells.
  • the rescued V L and the V H gene repertoires are spliced together and inserted into the minor coat protein of a bacteriophage (e.g., Ml 3 or fd, or a binding derivative thereof) to create a fusion bacteriophage coat protein (Chang et al, 1991, Expression of antibody Fab domains on bacteriophage surfaces. Potential use for antibody selection, J. Immunol.
  • the resulting bacteriophage contain a functional antibody fused to the outer surface of the phage protein coat and a copy of the gene fragment encoding the antibody V L and V H incorporated into the phage genome.
  • bacteriophage displaying antibodies that have affinity towards a particular antigen of interest can be isolated by, e.g., affinity chromatography, via the binding of a population of recombinant bacteriophage carrying the displayed antibody to a target epitope or antigen, which is immobilized on a solid surface or matrix. Repeated cycles of binding, removal of unbound or weakly-bound phage particles, and phage replication yield an enriched population of bacteriophage carrying the desired V L and V H gene fragments.
  • Antigens of interest may include peptides, proteins, immunoglobulin constant regions, CDRs (for production of anti-idiotypic antibodies) other macromolecules, haptens, small molecules, inorganic particles and surfaces.
  • CDRs for production of anti-idiotypic antibodies
  • the linked V and V H gene fragments can be rescued from the bacteriophage genome by standard DNA molecular techniques known in the art, cloned and expressed.
  • the number of antibodies created by this method is directly correlated to the size and diversity of the gene repertoire and offers an optimal method by which to create diverse antibody libraries that can be screened for antigenicity towards virtually any target molecule.
  • mAbs that have been created by antibody-phage-display technology often demonstrate specific binding towards antigen in the picomolar to nanomolar range (Sheets et al, 1998, Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens, Proc. Natl. Acad. Sci. USA 95(11): 6157-62).
  • Antibodies, or binding derivatives or binding fragments thereof, that are useful in the methods of the invention may be selected using an antibody or fragment phage display library constructed and characterized as described above.
  • Such an approach has the advantage of providing methods for efficiently screening a library having a high complexity (e.g. 10 9 ), so as to dramatically increase identification of antibodies or fragments suitable for use in the methods of the invention.
  • methods for cloning an immunoglobulin repertoire (“repertoire cloning") are used to produce an antibody for use in the staged-assembly methods of the invention. Repertoire cloning may be used for the production of virtually any kind of antibody without involving an antibody-producing animal.
  • mRNA messenger RNA
  • reverse transcriptase reverse transcriptase
  • Immunoglobulin cDNAs are specifically amplified by PCR, using the appropriate primers, from this complex mixture of cDNA.
  • PCR products from genes encoding antibody light (L) and heavy (H) chains are obtained. The products are then introduced into a phagemid vector.
  • Antigen-specific immunoglobulin is selected from this population by an iterative process of antigen immunoadsorption followed by phage multiplication.
  • a bacteriophage specific only for an antigen of interest will remain following multiple rounds of selection, and may be introduced into a new vector and/or host for further engineering or to express the phage-encoded protein in soluble form and in large amounts.
  • Antibody phage display libraries can thus be used, as described above, for the isolation, refinement, and improvement of epitope-binding regions of antibodies that can be used as joining elements in the construction of assembly units for use in the staged assembly of nanostructures, as disclosed herein.
  • Verification that two complementary joining elements interact with specificity may be established using, for example, ELISA assays, analytical ulfracentrifugation, or BIAcore methodologies (Abraham et al, 1996, Determination of binding constants of diabodies directed against prostate-specific antigen using electrochemiluminescence-based immunoassays, J. Mol. Recognit. 9(5-6): 456-61; Atwell et al, 1996, Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase, Mol. Immunol. 33(17-18): 1301-12; Muller et al.
  • a dimeric bispecific mini-antibody combines two specificities with avidity, FEBS Lett. 432(1-2): 45- 49), or other analogous methods well known in the art, that are suitable for demonstrating and/or quantitating the strength of intermolecular binding interactions.
  • Design of structural, joining and functional elements of the invention, and of the assembly units that comprise them, is facilitated by analysis and determination of those structures in the desired binding interaction, as revealed in a defined crystal structure, or through homology modeling based on a known crystal structure of a highly homologous material.
  • Design of a useful assembly unit comprising one or more functional elements preferably involves a series of decisions and analyses that may include, but are not limited to, some or all of the following steps:
  • nanoparticles such as quantum dots, etc., selection of structural elements that will provide a suitably rigid structure with correct dimensions and having positions for incorporation of joining elements with the correct geometry and stoichiometry; (v) design of fusion proteins incorporating peptide or protein j oining elements, from step (iii) and the structural element selected in step (iv) such that the folding of the structural and joining elements of the assembly unit are not disrupted (e.g., through incorporation at ⁇ -turns); (vi) computer modeling of the resultant fusion proteins in the context of the overall design of the nanostructure and refining of the design to optimize the structural dimensions as required by the functional specifications; or
  • Modification of a structural element protein usually involves insertion, deletion, or modification of the amino acid sequence of the protein in question. In many instances, modifications involve insertions or substitutions to add joining elements not extant in the native protein.
  • a non-limiting example of a routine test to determine the success of an insertion mutation is a circular dichroism (CD) spectrum.
  • the CD spectrum of the resultant fusion mutant protein can be compared to the CD of the native protein.
  • the spectra of a properly folded insertion mutant will be very similar to the spectra of the native protein. If the insertion is an entire protein domain (e.g. single chain variable domain), then the CD spectrum of the fusion protein should correspond to the sum of the CD spectra of the individual components (i.e. that of the native protein and fusion protein comprising the native protein and the functional element). This correspondence provides a routine test for the correct folding of the two components of the fusion protein.
  • a further test of the successful engineering of a fusion protein is made.
  • an analysis may be made of the ability of the fusion protein to bind to all of its targets, and therefore, to interact successfully with all joining pairs. This may be performed using a number of appropriate ELISA assays; at least one ELISA is perfomied to test the affinity and specificity of the modified protein for each of the joining pairs required to form the nanostructure.
  • the staged-assembly methods and the assembly units of the invention have use in the construction of myriad nanostructures.
  • the uses of such nanostructures are readily apparent and include applications that require highly regular, well-defined arrays of one-, two-, and three-dimensional structures such as fibers, cages, or solids, which may include specific attachment sites that allow them to associate with other materials.
  • the nanostructures fabricated by the staged assembly methods of the invention are one-dimensional structures.
  • nanostructures fabricated by staged assembly can be used for structural reinforcement of other materials, e.g., aerogels, paper, plastics, cement, etc.
  • nanostructures that are fabricated by staged assembly to take the form of long, one-dimensional fibers are incorporated, for example, into paper, cement or plastic during manufacture to provide added wet and dry tensile strength.
  • the nanostructure is a patterned or marked fiber that can be used for identification or recognition purposes.
  • the nanostructure may contain such functional elements as e.g., a fluorescent dye, a quantum dot, or an enzyme.
  • a particular nanostructure is impregnated into paper and fabric as an anti-counterfeiting marker.
  • a simple color-linked antibody reaction (such as those commercially available in kits) is used to verify the origin of the material.
  • a nanostructure could bind dyes, inks or other substances, either before or after incorporation, to color the paper or fabrics or to modify their appearance or properties in other ways.
  • nanostructures are incorporated, for example, into ink or dyes during manufacture to increase solubility or miscibility.
  • a one-dimensional nanostructure e.g., a fiber, bears one or more enzyme or catalyst functional elements in desired positions.
  • the nanostructure serves as a support structure or scaffold for an enzymatic or catalytic reaction to increase its efficiency.
  • the nanostructure may be used to "mount” or position enzymes or other catalysts in a desired reaction order to provide a reaction "assembly line.”
  • a one-dimensional nanostructure e.g., a fiber
  • Two or more components e.g., functional units, are bound to the nanostructure, thereby providing spatial orientation. The components are joined or fused, and then the resultant fused product is released from the nanostructure.
  • a nanostructure is a one-, two- or three-dimensional structure that is used as a support or framework for mounting nanoparticles (e.g., metallic or other particles with thermal, electronic or magnetic properties) with defined spacing, and is used to construct a nanowire or nanocircuit.
  • nanoparticles e.g., metallic or other particles with thermal, electronic or magnetic properties
  • the staged assembly methods of the invention are used to accomplish electrode-less plating of a one-dimensional nanostructure (fiber) with metal to construct a nanowire with a defined size and/or shape.
  • a nanostructure could be constructed that comprises metallic particles as functional elements.
  • a one-dimensional nanostructure (e.g., a fiber) comprising magnetic particles as functional elements is aligned by an external magnetic field to control fluid flow past the nanostructure.
  • the external magnetic field is used to align or dealign a nanostructure (e.g., fiber) comprising optical moieties as functional elements for use in LCD-type displays.
  • a nanostructure is used as a size standard or marker of precise dimensions for electron microscopy.
  • the nanostructures fabricated by the staged assembly methods of the invention are two- or three-dimensional structures.
  • the nanostructure is a mesh with defined pore size and can serve as a two- dimensional sieve or filter.
  • the nanostructure is a three-dimensional hexagonal array of assembly units that is employed as a molecular sieve or filter, providing regular vertical pores of precise diameter for selective separation of particles by size.
  • filters can be used for sterilization of solutions (i.e., to remove microorganisms or viruses), or as a series of molecular- weight cut-off filters,
  • the protein components of the pores, such as structural elements or functional elements may be modified so as to provide specific surface properties (i.e., hydrophilicity or hydrophobicity, ability to bind specific ligands, etc.).
  • specific surface properties i.e., hydrophilicity or hydrophobicity, ability to bind specific ligands, etc.
  • the methods and assembly units disclosed herein may be used to construct a variety of two- and three-dimensional structures such as polygonal structures (e.g., octagons), as well as open solids such as tetrahedrons, icosahedrons formed from triangles, and boxes or cubes formed from squares and rectangles (e.g., the cube disclosed in Section 11, Example 6).
  • polygonal structures e.g., octagons
  • open solids such as tetrahedrons, icosahedrons formed from triangles, and boxes or cubes formed from squares and rectangles (e.g., the cube disclosed in Section 11, Example 6).
  • the range of structures is limited only by the types of joining and functional elements that can be engineered on the different axes of the structural elements.
  • a two-or three-dimensional nanostructure may be used to construct a surface coating comprising optical, electric, magnetic, catalytic, or enzymatic moieties as functional units.
  • a coating could be used, for example, as an optical coating.
  • Such an optical coating could be used to alter the abso ⁇ tive or reflective properties of the material coated.
  • a surface coating constructed using nanostructures of the invention could also be used as an electrical coating, e.g., as a static shielding or a self-dusting surfaces for a lens (if the coating were optically clear). It could also be used as a magnetic coating, such as the coating on the surface of a computer hard drive.
  • Such a surface coating could also be used as a catalytic or enzymatic coating, for example, as surface protection.
  • the coating is an antioxidant coating.
  • the nanostructure may be used to construct an open framework or scaffold with optical, electric, magnetic, catalytic, enzymatic moieties as functional elements.
  • Such a scaffold may be used as a support for optical, electric, magnetic, catalytic, or enzymatic moieties as described above.
  • such a scaffold could comprise functional elements that are arrayed to form thicker or denser coatings of molecules, or to support soluble micron-sized particles with desired optical, electric, magnetic, catalytic, or enzymatic properties.
  • a nanostructure serves as a framework or scaffold upon which enzymatic or antibody binding domains could be linked to provide high density multivalent processing sites to link to and solubilize otherwise insoluble enzymes, or to entrap, protect and deliver a variety of molecular species.
  • the nanostructure may be used to construct a high density computer memory with addressable locations.
  • the nanostructure may be used to construct an artificial zeolite, i.e., a natural mineral (hydrous silicate) that has the capacity to absorb ions from water, wherein the design of the nanostructure promotes high efficiency processing with reactant flow-through an open framework.
  • the nanostructure may be used to construct an open framework or scaffold that serves as the basis for a new material, e.g., the framework may possess a unique congruency of properties such as strength, density, determinate particle packing and/or stability in various environments.
  • the staged-assembly methods of the invention can also be used for constructing computational architectures, such as quantum cellular automata (QCA) that are composed of spatially organized arrays of quantum dots.
  • QCA quantum cellular automata
  • the logic states are encoded by positions of individual electrons, contained in QCA cells composed of spatially positioned quantum dots, rather than by voltage levels.
  • Staged assembly can be implemented in an order that spatially organizes quantum dot particles in accordance with the geometries necessary for the storage of binary information.
  • Examples of logic devices that can be fabricated using staged assembly for the spatially positioning and construction of QCA cells for quantum dot cellular automata include QCA wires, QCA inverters, majority gates and full adders (Amlani et al, 1999, Digital logic gate using quantum-dot cellular automata, Science 284(5412): 289-91; Cowburn and Welland, 2000), Room temperature magnetic quantum cellular automata, Science 287(5457): 1466-68; Orlov et al, 1997, Realization of a Functional Cell for Quantum-Dot Automata, Science 277: 928-32).
  • This example discloses staged assembly using monovalent Fab fragments ("Fabl” and “Fab2,”) each with a different peptide epitope fused at their C-terminus (FIG. 4).
  • the CDR of Fabl has specificity for the peptide fused to the C-terminus of Fab2.
  • the CDR of Fab2 has specificity for the peptide fused to the C-terminus of Fabl.
  • the two joining pairs provide specific interactions between these two assembly units.
  • the first Fab can be immobilized to a solid substrate using standard methods. This surface can then be incubated with a solution containing Fab2 which has fused a peptide exhibiting specificity for Fabl. This incubation will result in the formation of a nanostructure intermediate comprised of one copy of Fabl (immobilized) and one copy of Fab2.
  • the intermediate can then be incubated against a solution containing Fabl, resulting in the formation of an intermediate comprised of a copy of Fabl attached to a copy of Fab2 that is sequentially attached to a copy of Fabl. This assembly process may then continue iteratively for as long as is necessary to achieve the size of linear structure required.
  • Assembly unit-1 is a monovalent assembly unit comprising an antibody Fab fragment with CDR (CDR1) that specifically binds to peptide 2 with a linked C-terminal peptide epitope (peptide 1).
  • Assembly unit-2 is a monovalent assembly unit comprising an antibody Fab fragment with CDR (CDR2) that specifically binds to peptide 1 with a linked C-terminal peptide epitope (peptide 2).
  • CDR2 antibody Fab fragment with CDR
  • Joining pair 1 Joining element peptide 1 interacts with joining element CDR 2.
  • Joining pair 2 Joining element peptide 2 interacts with joining element CDR 1.
  • Step 1 a) Add assembly unit-1 b) Wash
  • Step 4 a) Repeat Step 2 EXAMPLE 2 : STAGED ASSEMBLY USING MULTISPECIFIC PROTEIN
  • This example discloses an embodiment of the staged assembly methods of the invention that uses multispecific protein assembly units. Permutations and combinations of multispecific protein assembly units may be used for the construction of complex one-, two-, and three-dimensional macromolecular nanostructures, including, for example, the staged assembly illustrated in FIG. 14, which utilizes bivalent and tetravalent assembly units.
  • Staged assembly of a nanostructure comprising a four-point junction only requires a minimum of five assembly units and four joining pairs.
  • the five assembly units required include four bispecific and one tetraspecific assembly unit.
  • the joining pairs employed to join adjacent assembly units are idiotope/anti-idiotope in nature. A minimum of four such idiotope/anti-idiotope joining pairs are needed for staged-assembly in this example.
  • Assembly unit-1 is a bivalent protein assembly unit comprising a non-interacting (idiotope/anti-idiotope) joining pair A and B.
  • Assembly unit-2 is a bivalent assembly unit comprising a non-interacting (idiotope/anti-idiotope) joining pair B' and A'.
  • Assembly unit-3 is a tetravalent assembly unit comprising non-interacting (idiotope/anti-idiotope) joining pair B' and A' and non-interacting (idiotope/anti-idiotope) joining pair C and D.
  • Assembly unit-4 is a bivalent assembly unit comprising a non-interacting (idiotope/anti-idiotope) joining pair C and A.
  • Assembly unit-5 is a bivalent assembly unit with non-interacting (idiotope/anti-idiotope) joining pair D' and B'.
  • A interacts with A' in complementary joining pair 1.
  • B interacts with B' in complementary joining pair 2.
  • C interacts with C in complementary joining pair 3.
  • D interacts with D' in complementary joining pair 4.
  • FIG. 14 The following steps of staged assembly are illustrated in FIG. 14.
  • the resultant nanostructure is illustrated FIG. 14, Step 11.
  • Step 1 a) Add assembly unit-1 b) Wash
  • Step 5 a) Repeat Step 1
  • Step 7 a) Repeat Step 2
  • Step 9 a) Repeat Step 1
  • Step 11 a) Repeat Step 1 EXAMPLE 3 : FABRICATION OF A MACROMOLECUL AR
  • two assembled nanostructures intermediates can be joined to one another using the staged assembly methods of the invention.
  • This example describes the fabrication of a macromolecular nanostructure from two nanostructure intermediates.
  • FIG. 15 illustrates the staged assembly of the two nanostructure intermediates fabricated from the staged assembly protocol illustrated in FIG. 14.
  • Nanostructure intermediate- 1 is illustrated as Step-11 in FIG. 14.
  • Nanostructure intermediate-2 is illustrated as Step-8 in FIG. 14.
  • the protocol in Section 9J below describes the addition of two nanostructure intermediates by the association of a complementary joining pair.
  • FIG. 15 The following steps of staged assembly are illustrated in FIG. 15.
  • the resultant macromolecular nanostructure is illustrated FIG. 15, Step 5.
  • Step 4 Perform Steps 1-8 of staged assembly protocol described above in Section 8 (Example 3), leaving nanostructure intermediate-2 attached to the support matrix
  • Step 5 a) Add nanostructure intermediate- 1 b) Wash EXAMPLE 4: DEMONSTRATION OF SELF-ASSEMBLY AND STAGED
  • staged assembly may be carried out using two non-cross- reacting diabody assembly unit constructs that are expressed and purified. Solutions of each diabody unit protein alone should remain clear, since the single diabody assembly units will not self-polymerize (i.e., self-assemble).
  • the diabody units are capable of oligomerization as linked units and form long fibers in which the two diabody units alternate (FIG. 1).
  • This self-assembly is readily observable by eye, by simple light scattering or turbidity experiments and can be readily confirmed by electron microscopy of negatively stained polymer rods.
  • Staged assembly is carried out by immobilizing the initiator to a sepharose solid support matrix and then contacting the matrix-bound initiator with diabody assembly unit-1. This is followed by a wash step, in which excess diabody unit-1 is removed from the bound nanostructure (containing the initiator unit and bound diabody unit-1). The nanostructure is then incubated with diabody assembly unit-2, followed by washing and incubating in the presence of additional copies of diabody assembly unit-1, etc., through a number of cycles (FIG. 11). Electron microscopy is used to determine the length and geometry of the polymers assembled through different numbers of binding and wash cycles. These lengths are precisely proportional to the number of cycles.
  • the extent polymerization of macromolecular monomers may be analyzed by light scattering.
  • Light scattering measurements from a light scattering photometer e.g., the DAWN-DSP photometer (Wyatt Technology Co ⁇ ., Santa Barbara, CA), provides information for determination of the weight average molecular weight, determination of particle size, shape and particle-particle pair correlations.
  • EXAMPLE 6 MOLECULAR WEIGHT DETERMINATION (DEGREE OF POLYMERIZATION) BY SUCROSE GRADIENT SEDIMENTATION
  • Linked diabody units of different lengths sediment at different rates in a sucrose gradient in zonal ultracentrifugation.
  • the quantitative relationship between the degree of polymerization and sedimentation in Svedberg units is then calculated. This method is useful for characterizing the efficiency of self-assembly in general, as well as the process of staged assembly at each step of addition of a new diabody unit.
  • EXAMPLE 8 STAGED ASSEMBLY OF A THREE-DIMENSIONAL CUBE
  • This example discloses the fabrication of a three-dimensional cubic structure by staged assembly from assembly units comprising structural elements from engineered triabody and diabody fragments.
  • the joining elements of the assembly units are the multispecific binding domains of triabodies or diabodies.
  • Triabodies are trivalent and make up the vertices of the cubic-like structure.
  • Diabodies are bivalent and, in this example, two are used to construct the edges of the cubic structure, thereby spanning the space between the triabodies.
  • an added peptide epitope is engineered as a joining element within the triabody structural element for immobilization to a solid support (and defined as the first vertex of the cube in the staged assembly). Therefore the joining elements for the triabody initiator unit comprise four non-complementary joining elements, three of which are comprised of the trispecific binding domains of the triabody and the fourth from a peptide epitope engineered within the triabody structure designed specifically to interact with solid support matrix.
  • the peptide epitope comprised in the initiator unit can be engineered to contain a pre-designed releasing moiety (e.g.
  • a protease site that can be cleaved from the initiator unit and joined to the nanostructure from the solid support matrix upon complete nanofabrication of the three-dimensional nanocube. Since the three-dimensional structure of a triabody has been well characterized (Pei et al. , 1997, The 2.0-A resolution crystal structure of a trimeric antibody fragment with noncognate V H -V L domain pairs shows a rearrangement of V H CDR3, Proc. Natl. Acad. Sci. USA 94(18): 9637-42), the insertion points within the protein structure can be identified for engineering additional joining elements, as discussed hereinabove, by visual investigation of the available X-ray coordinates.
  • Another triabody comprised of three trispecific binding domains as the joining elements makes up another assembly unit (the other 7 vertices of the cube).
  • the other assembly units namely the diabody units comprised of two bispecific binding domains as joining elements, will form the edges of the cube (edges can be defined as the vectorial lattices between defined vertices of the cube).
  • Each edge of the cube will be fabricated from two diabody assembly units).
  • a total of 32 assembly units are required for the nanofabrication of a three-dimensional nanocube: 8 triabodies (one initiator unit and 7 assembly units making up the 8 vertices) and 24 diabodies (all assembly units making up the 12 edges).
  • Triabodies are three dimensional, equilateral triangle prism-shaped proteins that contain one joining element (CDR) at each of the three vertices.
  • Diabodies are rectangular prism shaped proteins with two opposing joining elements (CDRs).
  • CDR joining element
  • the nanofabrication of a three-dimensional (3-D) cube composed of triabodies and diabodies requires geometric and spatial relationships of the associated assembly units to be within defined design specifications of the three-dimensional cube shown in FIG. 16.
  • angles between adjacent Fab arms associated to the triabody was measured to be between 80-136° (i.e this falls within the required geometric and spatial relationships of the associated assembly units for the formation of a vertex associated with three edges of a cube) and that of a diabody and a Fab fragment associations was measured between 60 and 180° (this falls within the required geometric and spatial relationships for the formation of one edge of the cube upon the association (joining) of two adjacent diabody elements).
  • the angle between planar edges of the cube is defined as 90°and that of a cubic edge as 180°.
  • the cube is constructed by first identifying 7 non-cross-reacting, complementary joining element pairs, hi this embodiment, idiotope/anti-idiotope pairs are constructed using standard methods disclosed above.
  • the 14 joining elements that are elements of these pairs are inco ⁇ orated into bispecific diabodies and trispecific triabodies as indicated by the architecture disclosed below.
  • FIG. 16 is a diagram of the assembly of a cubic structure with the joining pairs indicated by letters (A being complementary to A'; B complementary to B', etc.); and the order of assembly indicated by numbers.
  • the first unit is the initiator unit, and it is indicated by the number T, and comprises joining elements A, B and C.
  • the second unit ('2') comprises joining elements A' and D.
  • Last added unit Joinir Lg elements exposed 1 A B C 2 D B C 3 A B C 4 A D C 5 A B C 6 A B D 7 A B C 8 E F B C 9 E D B C 10 E F B C 11 E F B A G 12 E F B D G 13 E F B A G 14 C E B G 15 C D B G 16 C E B G 17 C E A F G 18 C E D F G 19 C E A F G 20 C B F G 21 C D F G 22 C B F G 23 D B F G 24 C B F G 25 A F G 26 A F D 27 A F G 28 A D G

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Abstract

L'invention concerne des nanostructures comprenant au moins une espèce d'unité d'assemblage qui est une unité d'assemblage d'anticorps dans laquelle au moins deux éléments de jonction, un élément structural ou un élément fonctionnel est un anticorps ou un fragment d'anticorps. Les unités d'assemblage d'anticorps peuvent comporter plus d'un type d'élément anticorps. Elles peuvent aussi contenir des éléments de jonction, structuraux ou fonctionnels ne possédant pas les propriétés d'anticorps. La nanostructure est préparée correctement à l'aide d'un procédé d'assemblage en étapes. Un intermédiaire de nanostructure comportant au moins un élément de jonction non lié est mis en contact avec une unité d'assemblage comportant plusieurs éléments de jonction différents. Aucun de ces éléments de jonction de l'unité d'assemblage ne peut agir avec lui-même ou avec un autre élément de jonction de la même unité d'assemblage. Cependant, l'un des éléments de jonction de l'unité d'assemblage peut interagir avec l'élément de jonction non lié de l'intermédiaire de nanostructure, de façon que l'unité d'assemblage soit liée de façon non covalente à l'intermédiaire de nanostructure afin de former un nouvel intermédiaire de nanostructure à utiliser dans les cycles subséquents. Les unités d'assemblage non liées sont éliminées et le cycle est répété un nombre de fois suffisant afin de former une nanostructure. Les unités d'assemblage dans au moins un cycle sont des unités d'assemblage d'anticorps.
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KR20040102012A (ko) 2004-12-03
JP2005518455A (ja) 2005-06-23
WO2003072829A1 (fr) 2003-09-04
EP1485128A4 (fr) 2006-08-30
CA2477171A1 (fr) 2003-09-04
JP2005518454A (ja) 2005-06-23
CA2477271A1 (fr) 2003-09-04
EP1483408A4 (fr) 2005-06-22
AU2003217644A1 (en) 2003-09-09
KR20040094721A (ko) 2004-11-10
WO2003072804A3 (fr) 2004-03-25
AU2003215377A1 (en) 2003-09-09
WO2003072803A3 (fr) 2004-02-19
EP1485129A2 (fr) 2004-12-15
JP2005518456A (ja) 2005-06-23
US20030215903A1 (en) 2003-11-20
EP1483408A1 (fr) 2004-12-08
KR20040102011A (ko) 2004-12-03
EP1485129B1 (fr) 2007-01-10
WO2003072804A2 (fr) 2003-09-04
CA2477270A1 (fr) 2003-09-04
US20030198956A1 (en) 2003-10-23

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